Apoptosis related polynucleotides, polypeptides, and antibodies

ABSTRACT

The present invention relates to novel human apoptosis related polypeptides and isolated nucleic acids containing the coding regions of the genes encoding such polypeptides. Also provided are vectors, host cells, antibodies, and recombinant methods for producing human apoptosis related polypeptides. The invention further relates to diagnostic and therapeutic methods useful for diagnosing and treating disorders related to these novel human apoptosis related polypeptides.

[0001] This application is a continuation-in-part of, and claims benefitunder 35 U.S.C. §120 of copending PCT International Application SerialNo. PCT/US00/06642, filed Mar. 15, 2000, which is hereby incorporated byreference, which claims benefit under 35 U.S.C. §119(e) based on U.S.Provisional Applications No. 60/126,018, filed Mar. 24, 1999, No.60/139,638, filed Jun. 17, 1999, and No. 60/149,449, filed Aug. 18,1999, which are hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

[0002] The present invention relates to novel apoptosis relatedproteins. More specifically, isolated nucleic acid molecules areprovided encoding novel apoptosis related polypeptides. Novel apoptosisrelated polypeptides and antibodies that bind to these polypeptides areprovided. Also provided are vectors, host cells, and recombinant andsynthetic methods for producing human apoptosis related polynucleotidesand/or polypeptides. The invention further relates to diagnostic andtherapeutic methods useful for diagnosing, treating, preventing and/orprognosing disorders related to these novel apoptosis relatedpolypeptides. The invention further relates to screening methods foridentifying agonists and antagonists of polynucleotides and polypeptidesof the invention. The present invention further relates to methodsand/or compositions for inhibiting the production and function of thepolypeptides of the present invention.

BACKGROUND OF THE INVENTION

[0003] Apoptosis or programmed cell death is the innate mechanism bywhich the organism eliminates unwanted cells. In contrast to necrosis,apoptosis is the most common physiological form of cell death and occursduring embryonic development, tissue remodeling, immune regulation andtumor regression. Cells undergoing apoptosis show a sequence of cardinalmorphological features including membrane blebbing, cellular shrinkageand condensation of chromatin. Biochemically, these alterations areassociated with the translocation of phosphatidylserine to the outerleaflet of the plasma membrane and the activation of an endonucleasewhich cleaves genomic DNA into multiples of internucleosomal fragments.In contrast, necrosis is classically induced following traumatic injuryor exposure to high concentrations of noxious agents. Irreversibledamage of the plasma membrane, mitochondrial dysfunction and cell lysisare characteristic for necrotic cell death.

[0004] Higher organisms have developed several mechanisms to rapidly andselectively eliminate cells by apoptosis. A fine-tuned mechanism toregulate life and death of a cell is the interaction of surfacereceptors with their cognate ligands. Several receptors are able totransmit cytotoxic signals into the cytoplasm, but in most cases theyhave a wide range of other functions unrelated to cell death, such asinduction of cell activation, differentiation and proliferation. Whetherthe signals induced by a given receptor lead to cell activation or deathis highly cell-type specific and tightly regulated duringdifferentiation. For example, TNF receptors can exert co-stimulatorysignals for proliferation of naive lymphocytes as well as inducing deathsignals required for deletion of activated lymphocytes.

[0005] Many receptors with important functions in differentiation,survival and cell death belong to an emerging family of structurallyrelated molecules, called the TNF receptor superfamily. For some membersof the family an apoptosis-inducing activity has been reported. However,most of them also have other functions such as induction ofproliferation, differentiation, immune regulation and gene expression.Receptors with pleiotropic functions include TNF-R1, TNF-R2, NGF-R,CD27, CD30, CD40, OX-40, NGF-R, TRAMP (DR3/ws1-1/APO-3/LARD), HVEM(ATAR/TR2), GITR and RANK Anderson, D. M., et al., Nature 390, 175-179(1997); Bodmer, J. L. et al., Immunity 6, 79-88 (1997); Nocentini, G. etal., Proc. Natl. Acad. Sci. 94, 6216-6221 (1997)). These receptors aretype I membrane proteins which are structurally similar. Each possessesin its extracellular domain two-six imperfect repeats of about 40 aminoacids, with each of approximately six Cys residues. Their cytoplasmicdomains generally lack considerable sequence similarity.

[0006] APO-1/Fas, now called CD95, was the first member of the TNFreceptor superfamily described in terms of its function in apoptosis(Itoh, N. et al., Cell 66, 233-243 (1991); Oehm, A. et al., J. Biol.Chem. 267, 10709-10715 (1992)). Sequence comparison of the intracellulardomain of CD95 with TNF-R1 revealed that both receptors contained asimilar stretch of about 80 amino acids. This region has been designatedthe death domain (DD) since it enables transmission of a cytotoxicsignal by both molecules (Tartaglia, L. A. et al., Cell 74, 845-853(1993); Itoh, N. et al., J. Biol. Chem. 268, 10932-10937 (1993)). Recentsimilarity searches in EST databases led to the cloning of a number ofnovel membrane receptors that contain such a death domain and aretherefore referred to as the death receptors (DRs). TRAMP(DR3/ws1-1/APO-3/LARD), is both structurally and functionally similar toTNF-R1 and is abundantly expressed in T-lymphocytes (Bodmer, J. L. etal., Immunity 6, 79-88 (1997)). TRAIL-R1 (DR4, APO-2) and TRAIL-R2 (DR5)have been found as receptors binding to a novel cytokine, called TRAILfor TNF-related apoptosis-inducing ligand. The two TRAIL receptors arefunctionally similar to CD95 as their main function seems to be toinduce apoptosis (Pan, G. et al, Science 276, 111-113 (1997)). The TRAILsystem, in addition, consists of two neutralizing decoy receptors,called DcR1 (TRAIL-R3, TRID, LIT) and DcR2 (TRAIL-R4) (Pan, G. et al.,Science 277, 815-818 (1997); Degli-Esposti, M. A. et al., J. Exp. Med.186, 1165-1170 (1997)). The sequence of DcR1 encodes a protein thatcontains the external TRAIL-binding region as well as a stretch of aminoacids that anchors the receptor to the membrane. But, unlike the otherreceptors, DcR1 lacks an intracellular tail needed to spark the deathpathway. DcR2 is also able to bind TRAIL but contains a truncated deathdomain. Thus, both decoy receptors will prevent TRAIL from engagingfunctional TRAIL receptors and thereby render cells resistant toapoptosis. Collectively, this underlines that the death domain isrequired to induce apoptosis triggered by the different surfacereceptors.

[0007] For most members of the TNF-R superfamily their cognate ligandshave been identified. Four of them, CD95L, TNFα, lymphotoxin-α (LTα,TNFβ) and TRAIL bind to death receptors. It was not surprising to findthat, in addition to the receptors, also the ligands display structuralsimilarities, which are reflected by similar mechanisms of receptorrecognition and triggering. The ligands recognize their receptorsthrough a shared structure composed of anti-parallel β-sheets, arrangedin a jelly roll structure. As supported by structural and biochemicaldata, it is believed that all active ligands consist of three identicalsubunits and activate their receptors by oligomerization (Eck, M. J. etal., J. Biol.. Chem. 267, 2119-2122 (1992); Jones, E. Y., Immunol. Ser.56, 93-127 (1992); Banner, D. W. et al., Cell 73, 431-445 (1993); Dhein,J. et al., J. Immunol. 149, 3166-3173 (1992)). Another common feature ofthe ligands is that almost all of them are type II transmembraneproteins. The only exception is LTα which, although formed as a solubleprotein, binds to membrane-bound LTβ and thereby also acts as acell-bound form. Lymphotoxins can be found as homotrimers (LTα₃) orheterotrimers (LTα₁/β₂ or LTα₂/β₁). The LTα homotrimer binds the TNFreceptors, whereas the heterotrimers bind to the LTβ receptor which doesnot contain a death domain. Although TNF-related ligands are synthesizedas membrane-bound molecules, most of them also exist as soluble forms.The secreted forms are generated by rather specific metalloproteases.For TNF, a zinc-dependent metalloprotease, called TACE (TNFα-convertingenzyme) was recently cloned and shown to specifically cleave TNF (Black,R. A. et al., Nature 385, 729-733 (1997); Moss, M. L. et al., Nature385, 733-736 (1997)).

[0008] Death Receptor-associating Proteins

[0009] A major progress in the understanding of death receptor signalingwas the definition of the so-called death domain (DD), an intracellularregion of about 80 amino acids that is essential for triggering celldeath. Delineation of the DD was not only a major aid for theidentification of new adaptor molecules when used as a bait ininteractive cloning approaches. The DD exerts its effects viainteractive properties, as it can self-associate and bind to the DD ofother proteins. These associations between DDs occur as a consequence ofreceptor-ligand binding and seem to involve electrostatic interactions.As assessed by NMR spectroscopy, the DD of CD95 comprises a series ofantiparallel amphipathic α-helices with many exposed charged residues(Huang, B. et al., Nature 384, 638-641 (1996)), although it should benoted that this structure was determined at acidic pH. The tendency ofthe DD to self-associate apparently strengthens the interactions of thereceptors imposed by ligand binding. Following self-association, the DDof the receptors recruits other DD-containing proteins which then serveas adapters in the signaling cascades.

[0010] The first DD-containing adaptor proteins identified were FADD(MORT1) (Chinnaiyan et al., Cell, 81:505-512 (1995); Boldin et al., J.Biol. Chem., 270:7795-7798 (1995)), RIP (Stanger et al., Cell,81:513-523 (1995)) and TRADD (Hsu et al., Cell, 81:495-504 (1995)).TRADD is most effectively bound following ligation of TNF-R1 where itthen probably serves to recruit the DD proteins FADD and RIP as well asthe RING domain adaptor protein TRAF2, FADD, in contrast, ispreferentially recruited to CD95. Thus, the DD of FADD can bind to theDD of TRADD and the DD of RIP to the DDs of both TRADD and FADD. Thesemutual interactions may account for a potential crosstalk of thedifferent receptor signaling pathways.

[0011] Overexpression of most DD proteins causes cell death, indicatingthat these molecules are involved in apoptosis signaling. In the case ofFADD, transient expression of the N-terminal region was sufficient tocause apoptosis. (Chinnaiyan et al., Cell, 81:505-512 (1995)). This partof FADD was therefore termed the death effector domain (DED). Incontrast, overexpression of the C-terminal DD-containing part, lackingthe DED (FADD-DN), protected cells from CD95-mediated apoptosis andfunctioned as a dominant-negative mutant. This suggested that theN-terminus of FADD is coupled to the cytotoxic machinery. Both TRADD andRIP induce apoptosis but can also activate NF-κB, which is a typicalfeature of TNF-induced signaling. (Hsu et al., Cell, 81:495-504 (1995)).Similar to FADD, the C-terminus of TRADD contains a DD enablingself-association and association with the DD of other signalingmolecules including TNF-R1 and FADD. TRADD, however, lacks the typicalDED present in FADD.

[0012] While most of the information regarding death pathways has beenobtained from yeast two-hybrid assays or supra-physiologicaloverexpression in mammalian cells, for CD95 the signaling complexes havealso been identified in vivo using classical biochemical methods.(Kischkel et al., EMBO J., 14:5579-5588 (1995)). Treatment of cells withagonistic anti-APO-1 antibodies and subsequent co-immunoprecipitation ofCD95 resulted in the identification of four cytotoxicity-dependentAPO-1-associated proteins (CAP1-4) on two-dimensional gels, withinseconds after receptor triggering. Together with the receptor, theseproteins formed the death-inducing signaling complex (DISC). Two spotswere identified as two different serine-phosphorylated species of FADD,and it was demonstrated that FADD bound to CD95 in astimulation-dependent fashion.

[0013] Sequencing of the other immunoprecipitated proteins resulted inthe identification of a downstream molecule which contained two DEDs atits N-terminus that associate with the DED of FADD. (Muzio et al., Cell,85:817-827 (1996)). At its C-terminus it had the typical domainstructure of a protease like interleukin-1β converting enzyme (ICE) andwas therefore termed FLICE (FADD-like ICE). FLICE was also cloned by twoother groups and named MACH and Mch5. (Srinivasula et al., Proc. Natl.Acad. Sci. USA , 93:14486-14491 (1996); Alnemri et al., Cell, 87:171(1996)). It belongs to cysteine proteases of the caspase family and istherefore now referred to as caspase-8 (Alnemri. et al., Cell, 87:171(1996)).

[0014] Caspases have been found in organisms ranging from C. elegans tohumans. Caspases were implicated in apoptosis with the discovery thatCED-3, the product of a gene required for cell death in the nematodeCaenorhabditis elegans, is related to mammalianinterleukin-1[beta]-converting enzyme (ICE or caspase-1). (S. Yuan etal., Cell, 75:641 (1993); Thornberry et al., Nature, 356:768 (1992)).Although caspase-1 has no obvious role in cell death, it has become thefirst identified member of a large family of proteases whose membershave distinct roles in inflammation and apoptosis. In apoptosis,caspases function in both cell disassembly (effectors) and in initiatingthis disassembly in response to proapoptotic signals (initiators).

[0015] Caspases share similarities in amino acid sequence, structure,and substrate specificity. (Nicholson et al., Trends Biochem. Sci.,22:299 (1997)). They are all expressed as proenzymes (30 to 50 kD) thatcontain three domains: an NH2-terminal domain, a large subunit (˜20 kD),and a small subunit (˜10 kD). Activation involves proteolytic processingbetween domains, followed by association of the large and small subunitsto form a heterodimer. Crystal structures of two active caspases(caspase-1 and caspase-3) have been determined: in both cases, twoheterodimers associate to form a tetramer, with two catalytic sites thatappear to function independently. (Walker et al., Cell, 78:343 (1994);Wilson et al., Nature, 370:270 (1994); Rotonda et al., Nature Struct.Biol., 3:619 (1996)). Within each catalytic domain, the large and smallsubunits are intimately associated, with both contributing residuesnecessary for substrate binding and catalysis.

[0016] Two features of the proenzyme structure are central to themechanism of activation of these enzymes. First, the NH2-terminaldomain, which is highly variable in sequence and length, is involved inregulation of activation. Second, all domains are derived from theproenzyme by cleavage at caspase consensus sites, implying that theseenzymes can be activated either autocatalytically or in a cascade byenzymes with similar specificity.

[0017] Caspases are among the most specific of proteases, with anunusual and absolute requirement for cleavage after aspartic acid (Theonly other eukaryotic protease known to have a similar specificity isthe serine protease granzyme B, a mediator of granule-dependentcytotoxic T lymphocyte-mediated apoptosis). Recognition of at least fouramino acids NH2-terminal to the cleavage site is also a necessaryrequirement for efficient catalysis. The preferred tetrapeptiderecognition motif differs significantly among caspases and explains thediversity of their biological functions. (Thornberry et al., J. Biol.Chem., 272:17907 (1997)). Their specificity is even more stringent: notall proteins that contain the optimal tetrapeptide sequence are cleaved,implying that tertiary structural elements may influence substraterecognition. Cleavage of proteins by caspases is not only specific, butalso highly efficient (k_(cat)/K_(m)>10⁶ M⁻¹ s⁻¹). The strictspecificity of caspases is consistent with the observation thatapoptosis is not accompanied by indiscriminate protein digestion;rather, a select set of proteins is cleaved in a coordinated manner,usually at a single site, resulting in a loss or change in function.

[0018] As stated, apoptotic events include DNA fragmentation, chromatincondensation, membrane blebbing, cell shrinkage, and disassembly intomembrane-enclosed vesicles (apoptotic bodies). In vivo, this processculminates with the engulfment of apoptotic bodies by other cells,preventing complications that would result from a release ofintracellular contents. These changes occur in a predictable,reproducible sequence and can be completed within 30 to 60 min. Currentresearch suggests that a subset of caspases (effectors) is responsiblefor the cellular changes that occur during apoptosis and provideinsights into the mechanisms that they employ.

[0019] One role of caspases is to inactivate proteins that protectliving cells from apoptosis. A clear example is the cleavage ofICAD/DFF45 (Enari et al., Nature, 391:43 (1998); Liu et al., Cell,89:175 (1997)), an inhibitor of the nuclease responsible for DNAfragmentation, CAD (caspase-activated deoxyribonuclease). Innonapoptotic cells, CAD is present as an inactive complex with ICAD.During apoptosis, ICAD is inactivated by caspases, leaving CAD free tofunction as a nuclease. This system is not as simple as it appears: CADsynthesized in the absence of ICAD is not active, implying that theCAD-ICAD complex is formed co-translationally, and that ICAD is requiredfor both the activity and inhibition of this nuclease.

[0020] Other negative regulators of apoptosis cleaved by caspases areBcl-2 proteins. (Xue et al., Nature, 390:305 (1997); Cheng et al.,Science, 278:1966 (1997); Cory ibid. 281:1322 (1998)). It appears thatcleavage not only inactivates these proteins, but also produces afragment that promotes apoptosis. That such positive feedbacks areinvolved in the control of apoptosis is not surprising, given theirimportance in the regulation of other proteolytic systems.

[0021] Caspases contribute to apoptosis through direct disassembly ofcell structures, as illustrated by the destruction of nuclear lamina(Takahashi et al., Proc. Natl. Acad. Sci. U.S.A., 93:8395 (1996); Orthet al., J. Biol. Chem., 271:16443 (1996)), a rigid structure thatunderlies the nuclear membrane and is involved in chromatinorganization. Lamina is formed by head-to-tail polymers of intermediatefilament proteins called lamins. During apoptosis, lamins are cleaved ata single site by caspases, causing lamina to collapse and contributingto chromatin condensation.

[0022] Caspases also reorganize cell structures indirectly by cleavingseveral proteins involved in cytoskeleton regulation, including gelsolin(S. Kothakota et al., Science, 278:294 (1997)), focal adhesion kinase(FAK) (Wen et al., J. Biol. Chem. 272:26056 (1997)), and p21-activatedkinase 2 (PAK2). Cleavage of these proteins results in deregulation oftheir activity. For example, in the case of gelsolin (a protein thatsevers actin filaments in a regulated manner), caspase cleavagegenerates a fragment that is instead constitutively active.

[0023] Dissociation of regulatory and effector domains is a hallmark ofcaspase function. For example, they inactivate or deregulate proteinsinvolved in DNA repair (such as DNA-PKcs), mRNA splicing (such asU1-70K), and DNA replication (such as replication factor C). Althoughthe relationship of these cleavages to cell death is not clearlyunderstood, it is likely that the disabling of critical homeostatic andrepair functions facilitates cellular disassembly.

[0024] The observations that caspase precursors are constitutivelyexpressed in living cells (even in neurons that can live for a lifetime)but that apoptosis can be induced quickly indicates that caspaseregulation is sophisticated and effective. Complex proteolytic systemsoften involve a combination of regulatory proteases, cofactors,feedbacks, and thresholds that converge to control the activity of aneffector protease, that in turn carries out the function of the wholeprocess. (Beltrami et al., Proc. Natl. Acad. Sci. U.S.A., 92:8744(1995)). This intricate regulation accounts for a spectacular feature ofthese systems: they keep the effector protease inactive but are able torapidly activate large amounts of it in response to minute quantities ofan appropriate inducer. Given the function of caspases as mediators ofcell death, the complexity of their regulation is likely to rival thatof the coagulation and complement systems.

[0025] Activation of effector caspases. A large body of genetic andbiochemical evidence supports a cascade model for effector caspaseactivation: a proapoptotic signal culminates in activation of aninitiator caspase which, in turn, activates effector caspases, resultingin cellular disassembly. Different initiator caspases mediate distinctsets of signals. For example, caspase-8 is associated with apoptosisinvolving death receptors. (Ashkenazi et al., Science, 281:1305 (1998)).In contrast, caspase-9 is involved in death induced by cytotoxic agents.(Hakem et al., Cell, in press; Kuida et al., ibid., in press). Thismodel explains how distinct apoptotic signals induce the samebiochemical and morphological changes.

[0026] Activation of initiator caspases. The available evidenceindicates that activation of initiator caspases requires binding tospecific cofactors, a mechanism commonly observed with proteases. Thisbinding is triggered by a proapoptotic signal and mediated through oneof at least two distinct structural motifs that reside in both thecaspase prodomain and its corresponding cofactor. Activation ofprocaspase-8 requires association with its cofactor FADD (Fas-associatedprotein with death domain) through the DED (death effector domain)Boldin et al. ibid., 85:803 (1996); Muzio et al., ibid., p. 817), whileprocaspase-9 activation involves a complex with the cofactor APAF-1through the CARD (caspase recruitment domain) (Li et al., ibid., 91:479(1997)). Activation of caspase-9 also requires cytochrome c anddeoxyadenosine triphosphate, indicating that caspase activation mayrequire multiple cofactors.

[0027] Thus there exists a clear need for identifying and exploitingnovel apoptosis related proteins. Although structurally related, suchproteins may possess diverse and multifaceted functions in a variety ofcell and tissue types. The inventive purified apoptosis relatedpolypeptides are research tools useful for the identification,characterization and purification of additional proteins involved inapoptosis. Furthermore, the identification of new apoptosis relatedpolypeptides permits the development of a range of derivatives, agonistsand antagonists at the nucleic acid and protein levels which in turnhave applications in the treatment and diagnosis of a range ofconditions such as cancer, inflammation, neurological disorders andaberrant cell growth, amongst many other conditions.

SUMMARY OF THE INVENTION

[0028] The present invention includes isolated nucleic acid moleculescomprising, or alternatively, consisting of a polynucleotide sequencedisclosed in the sequence listing and/or contained in a human cDNAplasmid described in Table 1 and deposited with the American TypeCulture Collection (ATCC). Fragments, variants, and derivatives of thesenucleic acid molecules are also encompassed by the invention. Thepresent invention also includes isolated nucleic acid moleculescomprising, or alternatively, consisting of, a polynucleotide encodingapoptosis related polypeptides. The present invention further includesapoptosis related polypeptides encoded by these polynucleotides. Furtherprovided for are amino acid sequences comprising, or alternatively,consisting of, apoptosis related polypeptides as disclosed in thesequence listing and/or encoded by the human cDNA plasmids described inTable 1 and deposited with the ATCC. Antibodies that bind thesepolypeptides are also encompassed by the invention. Polypeptidefragments, variants, and derivatives of these amino acid sequences arealso encompassed by the invention, as are polynucleotides encoding thesepolypeptides and antibodies that bind these polypeptides.

DETAILED DESCRIPTION

[0029] Tables

[0030] Table 1 summarizes ATCC Deposits, Deposit dates, and ATCCdesignation numbers of deposits made with the ATCC in connection withthe present application. Table 1 further summarizes the informationpertaining to each “Gene No.” described below, including cDNA cloneidentifier, the type of vector contained in the cDNA clone identifier,the nucleotide sequence identifier number, nucleotides contained in thedisclosed sequence, the location of the 5′ nucleotide of the start codonof the disclosed sequence, the amino acid sequence identifier number,and the last amino acid of the ORF encoded by the disclosed sequence.

[0031] Table 2 indicates public ESTs, of which at least one, two, three,four, five, ten, or more of any one or more of these public ESTsequences are optionally excluded from certain embodiments of theinvention.

[0032] Table 3 summarizes the expression profile of polynucleotidescorresponding to the clones disclosed in Table 1. The first columnprovides a unique clone identifier, “Clone ID NO:Z”, for a cDNA clonerelated to each contig sequence disclosed in Table 1. Column 2, “LibraryCode” shows the expression profile of tissue and/or cell line librarieswhich express the polynucleotides of the invention. Each Library Code incolumn 2 represents a tissue/cell source identifier code correspondingto the Library Code and Library description provided in Table 5.Expression of these polynucleotides was not observed in the othertissues and/or cell libraries tested. One of skill in the art couldroutinely use this information to identify tissues which show apredominant expression pattern of the corresponding polynucleotide ofthe invention or to identify polynucleotides which show predominantand/or specific tissue expression.

[0033] Table 4, column 1, provides a nucleotide sequence identifier,“SEQ ID NO:X,” that matches a nucleotide SEQ ID NO:X disclosed in Table1, column 5. Table 4, column 2, provides the chromosomal location,“Cytologic Band or Chromosome,” of polynucleotides corresponding to SEQID NO:X. Chromosomal location was determined by finding exact matches toEST and cDNA sequences contained in the NCBI (National Center forBiotechnology Information) UniGene database. Given a presumptivechromosomal location, disease locus association was determined bycomparison with the Morbid Map, derived from Online MendelianInheritance in Man (Online Mendelian Inheritance in Man, OMIM™.McKusick-Nathans Institute for Genetic Medicine, Johns HopkinsUniversity (Baltimore, Md.) and National Center for BiotechnologyInformation, National Library of Medicine (Bethesda, Md.) 2000. WorldWide Web URL: http://www.ncbi.nlm.nih.gov/omim/). If the putativechromosomal location of the Query overlapped with the chromosomallocation of a Morbid Map entry, the OMIM reference identification numberof the morbid map entry is provided in Table 4, column 3, labelled “OMIMID.” A key to the OMIM reference identification numbers is provided inTable 6.

[0034] Table 5, column 1, provides the Library Code disclosed in Table3, column 2. Column 2 provides a description of the tissue or cellsource from which the corresponding library was derived.

[0035] Table 6 provides a key to the OMIM reference identificationnumbers disclosed in Table 4, column 3. OMIM reference identificationnumbers (Column 1) were derived from Online Mendelian Inheritance in Man(Online Mendelian Inheritance in Man, OMIM. McKusick-Nathans Institutefor Genetic Medicine, Johns Hopkins University (Baltimore, Md.) andNational Center for Biotechnology Information, National Library ofMedicine, (Bethesda, Md.) 2000. World Wide Web URL:http://www.ncbi.nlm.nih.gov/omim/). Column 2 provides diseasesassociated with the cytologic band disclosed in Table 4, column 2, asdetermined from the Morbid Map database.

[0036] Definitions

[0037] The following definitions are provided to facilitateunderstanding of certain terms used throughout this specification.

[0038] In the present invention, “isolated” refers to material removedfrom its original environment (e.g., the natural environment if it isnaturally occurring), and thus is altered “by the hand of man” from itsnatural state. For example, an isolated polynucleotide could be part ofa vector or a composition of matter, or could be contained within acell, and still be “isolated” because that vector, composition ofmatter, or particular cell is not the original environment of thepolynucleotide. The term “isolated” does not refer to genomic or cDNAlibraries, whole cell total or mRNA preparations, genomic DNApreparations (including those separated by electrophoresis andtransferred onto blots), sheared whole cell genomic DNA preparations orother compositions where the art demonstrates no distinguishing featuresof the polynucleotide/sequences of the present invention.

[0039] As used herein, a “polynucleotide” refers to a molecule having anucleic acid sequence contained in SEQ ID NO:X (as described in column 5of Table 1), or cDNA plasmid:Z (as described in column 3 of Table 1 andcontained within a pool of plasmids deposited with the ATCC). Forexample, the polynucleotide can contain the nucleotide sequence of thefull length cDNA sequence, including the 5′ and 3′ untranslatedsequences, the coding region, with or without a natural or artificialsignal sequence, the protein coding region, as well as fragments,epitopes, domains, and variants of the nucleic acid sequence. Moreover,as used herein, a “polypeptide” refers to a molecule having an aminoacid sequence encoded by a polynucleotide of the invention as broadlydefined (obviously excluding poly-Phenylalanine or poly-Lysine peptidesequences which result from translation of a polyA tail of a sequencecorresponding to a cDNA).

[0040] In the present invention, a representative plasmid containing thesequence of SEQ ID NO:X was deposited with the American Type CultureCollection (“ATCC”) and/or described in Table 1. As shown in Table 1,each plasmid is identified by a cDNA Clone ID (Identifier) and the ATCCDeposit Number (ATCC Deposit No:Z). Plasmids that were pooled anddeposited as a single deposit have the same ATCC Deposit Number. TheATCC is located at 10801 University Boulevard, Manassas, Va. 20110-2209,USA. The ATCC deposit was made pursuant to the terms of the BudapestTreaty on the international recognition of the deposit of microorganismsfor purposes of patent procedure.

[0041] A “polynucleotide” of the present invention also includes thosepolynucleotides capable of hybridizing, under stringent hybridizationconditions, to sequences contained in SEQ ID NO:X, or the complementthereof (e.g., the complement of any one, two, three, four, or more ofthe polynucleotide fragments described herein) and/or sequencescontained in cDNA plasmid:Z (e.g., the complement of any one, two,three, four, or more of the polynucleotide fragments described herein).“Stringent hybridization conditions” refers to an overnight incubationat 42 degree C. in a solution comprising 50% formamide, 5×SSC (750 mMNaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6),5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured,sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC atabout 65 degree C.

[0042] Also included within “polynucleotides” of the present inventionare nucleic acid molecules that hybridize to the polynucleotides of thepresent invention at lower stringency hybridization conditions. Changesin the stringency of hybridization and signal detection are primarilyaccomplished through the manipulation of formamide concentration (lowerpercentages of formamide result in lowered stringency); salt conditions,or temperature. For example, lower stringency conditions include anovernight incubation at 37 degree C. in a solution comprising 6×SSPE(20×SSPE=3M NaCl; 0.2M NaH₂PO₄; 0.02M EDTA, pH 7.4), 0.5% SDS, 30%formamide, 100 ug/ml salmon sperm blocking DNA; followed by washes at 50degree C. with 1×SSPE, 0.1% SDS. In addition, to achieve even lowerstringency, washes performed following stringent hybridization can bedone at higher salt concentrations (e.g. 5×SSC).

[0043] Note that variations in the above conditions may be accomplishedthrough the inclusion and/or substitution of alternate blocking reagentsused to suppress background in hybridization experiments. Typicalblocking reagents include Denhardt's reagent, BLOTTO, heparin, denaturedsalmon sperm DNA, and commercially available proprietary formulations.The inclusion of specific blocking reagents may require modification ofthe hybridization conditions described above, due to problems withcompatibility.

[0044] Of course, a polynucleotide which hybridizes only to polyA+sequences (such as any 3′ terminal polyA+ tract of a cDNA shown in thesequence listing), or to a complementary stretch of T (or U) residues,would not be included in the definition of “polynucleotide,” since sucha polynucleotide would hybridize to any nucleic acid molecule containinga poly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone generated using oligo dT as a primer).

[0045] The polynucleotides of the present invention can be composed ofany polyribonucleotide or polydeoxribonucleotide, which may beunmodified RNA or DNA or modified RNA or DNA. For example,polynucleotides can be composed of single- and double-stranded DNA, DNAthat is a mixture of single- and double-stranded regions, single- anddouble-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded or a mixtureof single- and double-stranded regions. In addition, the polynucleotidecan be composed of triple-stranded regions comprising RNA or DNA or bothRNA and DNA. A polynucleotide may also contain one or more modifiedbases or DNA or RNA backbones modified for stability or for otherreasons. “Modified” bases include, for example, tritylated bases andunusual bases such as inosine. A variety of modifications can be made toDNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically,or metabolically modified forms.

[0046] In specific embodiments, the polynucleotides of the invention areat least 15, at least 30, at least 50, at least 100, at least 125, atleast 500, or at least 1000 continuous nucleotides but are less than orequal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5kb, 5 kb, 2.5kb, 2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotidesof the invention comprise a portion of the coding sequences, asdisclosed herein, but do not comprise all or a portion of any intron. Inanother embodiment, the polynucleotides comprising coding sequences donot contain coding sequences of a genomic flanking gene (i.e., 5′ or 3′to the gene of interest in the genome). In other embodiments, thepolynucleotides of the invention do not contain the coding sequence ofmore than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1genomic flanking gene(s).

[0047] “SEQ ID NO:X” refers to a polynucleotide sequence described incolumn 5 of Table 1, while “SEQ ID NO:Y” refers to a polypeptidesequence described in column 10 of Table 1. SEQ ID NO:X is identified byan integer specified in column 6 of Table 1. The polypeptide sequenceSEQ ID NO:Y is a translated open reading frame (ORF) encoded bypolynucleotide SEQ ID NO:X. The polynucleotide sequences are shown inthe sequence listing immediately followed by all of the polypeptidesequences. Thus, a polypeptide sequence corresponding to polynucleotidesequence SEQ ID NO:2 is the first polypeptide sequence shown in thesequence listing. The second polypeptide sequence corresponds to thepolynucleotide sequence shown as SEQ ID NO:3, and so on.

[0048] The polypeptides of the present invention can be composed ofamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres, and may contain amino acids other thanthe 20 gene-encoded amino acids. The polypeptides may be modified byeither natural processes, such as posttranslational processing, or bychemical modification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched, for example, as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic polypeptides may result fromposttranslation natural processes or may be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. (See, forinstance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993); POSTTRANSLATIONALCOVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press,New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646(1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992)).

[0049] The polypeptides of the invention can be prepared in any suitablemanner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

[0050] The polypeptides may be in the form of the secreted protein,including the mature form, or may be a part of a larger protein, such asa fusion protein (see below). It is often advantageous to include anadditional amino acid sequence which contains secretory or leadersequences, pro-sequences, sequences which aid in purification, such asmultiple histidine residues, or an additional sequence for stabilityduring recombinant production.

[0051] The polypeptides of the present invention are preferably providedin an isolated form, and preferably are substantially purified. Arecombinantly produced version of a polypeptide, including the secretedpolypeptide, can be substantially purified using techniques describedherein or otherwise known in the art, such as, for example, by theone-step method described in Smith and Johnson, Gene 67:31-40 (1988).Polypeptides of the invention also can be purified from natural,synthetic or recombinant sources using techniques described herein orotherwise known in the art, such as, for example, antibodies of theinvention raised against the polypeptides of the present invention inmethods which are well known in the art.

[0052] By a polypeptide demonstrating a “functional activity” is meant,a polypeptide capable of displaying one or more known functionalactivities associated with a full-length (complete) protein of theinvention. Such functional activities include, but are not limited to,biological activity, antigenicity [ability to bind (or compete with apolypeptide for binding) to an anti-polypeptide antibody],inmunogenicity (ability to generate antibody which binds to a specificpolypeptide of the invention), ability to form multimers withpolypeptides of the invention, and ability to bind to a receptor orligand for a polypeptide. “A polypeptide having functional activity”refers to polypeptides exhibiting activity similar, but not necessarilyidentical to, an activity of a polypeptide of the present invention,including mature forms, as measured in a particular assay, such as, forexample, a biological assay, with or without dose dependency. In thecase where dose dependency does exist, it need not be identical to thatof the polypeptide, but rather substantially similar to thedose-dependence in a given activity as compared to the polypeptide ofthe present invention (i.e., the candidate polypeptide will exhibitgreater activity or not more than about 25-fold less and, preferably,not more than about tenfold less activity, and most preferably, not morethan about three-fold less activity relative to the polypeptide of thepresent invention).

[0053] The functional activity of the polypeptides, and fragments,variants derivatives, and analogs thereof, can be assayed by variousmethods.

[0054] For example, in one embodiment where one is assaying for theability to bind or compete with full-length polypeptide of the presentinvention for binding to an antibody to the full length polypeptide,various immnunoassays known in the art can be used, including but notlimited to, competitive and non-competitive assay systems usingtechniques such as radioinmmunoassays, ELISA (enzyme linkedimmunosorbent assay), “sandwich” inmmunoassays, immunoradiometricassays, gel diffusion precipitation reactions, immunodiffusion assays,in situ immunoassays (using colloidal gold, enzyme or radioisotopelabels, for example), western blots, precipitation reactions,agglutination assays (e.g., gel agglutination assays, hemagglutinationassays), complement fixation assays, immunofluorescence assays, proteinA assays, and inmmunoelectrophoresis assays, etc. In one embodiment,antibody binding is detected by detecting a label on the primaryantibody. In another embodiment, the primary antibody is detected bydetecting binding of a secondary antibody or reagent to the primaryantibody. In a further embodiment, the secondary antibody is labeled.Many means are known in the art for detecting binding in an immunoassayand are within the scope of the present invention.

[0055] In another embodiment, where a ligand is identified, or theability of a polypeptide fragment, variant or derivative of theinvention to multimerize is being evaluated, binding can be assayed,e.g., by means well-known in the art, such as, for example, reducing andnon-reducing gel chromatography, protein affinity chromatography, andaffinity blotting. See generally, Phizicky, E., et al., Microbiol. Rev.59:94-123 (1995). In another embodiment, physiological correlatespolypeptide of the present invention binding to its substrates (signaltransduction) can be assayed.

[0056] In addition, assays described herein (see Examples) and otherwiseknown in the art may routinely be applied to measure the ability ofpolypeptides of the present invention and fragments, variantsderivatives and analogs thereof to elicit polypeptide related biologicalactivity (either in vitro or in vivo). Other methods will be known tothe skilled artisan and are within the scope of the invention.

[0057] Polynucleotides and Polypeptides of the Invention

[0058] Features of Protein Encoding by Gene No: 1

[0059] Translation products corresponding to this gene share sequencehomology with the human death effector domain-containing molecule, DEDD(See Genbank Accession CAA09445), which is thought to play a role ininhibiting transcription within cells. Several proteins contain a domaincalled a death effector domain (DED). These DED domains interact withother proteins in the apoptotic pathway to either inhibit or promoteapoptosis. The pivotal protein in this pathway, FADD, contains both aDeath-domain (DD) and a DED. Although the death domain is required forit to associate with Fas and to initiate apoptosis, it is not sufficientfor apoptosis. In fact, the expression of only the death domain inhibitsapoptosis. The DED is the critical part of the molecule, recruitingdownstream proteins that actually effect apoptosis. Overexpression ofthis domain alone is sufficient for apoptosis. The DED of FADD recruits,through their own death effector domains, caspase-8 and caspase-10,which are the most proximal and critical caspases in the death signalingpathway. Overexpression of the death domain alone inhibits apoptosissince it occupies Fas but does not allow association of the caspases,and possibly other critical molecules. Several death effector-containingproteins have been discovered that function completely opposite FADD toinhibit apoptosis. The DED of equine herpesvirus protein E8 interactswith the caspase-8 prodomain whereas that in molluscum contagiosum virusprotein MC159 interacts with FADD. Both of these interactions block bothFas- and TNFR1-induced apoptosis. Binding of MC159 with FADD presumablyprevents FADD from recruiting the caspase-8 and/or -10. E8 may inhibitcaspase-8 directly by binding to it.

[0060] In specific embodiments, polypeptides of the invention comprise,or alternatively consist of, the following amino acid sequence:PGSTHASGKIQNKWLRPSPRSHRTPESGRVLSLFRLPPPG (SEQ ID NO: 20).Polynucleotides encoding these polypeptides are also encompassed by theinvention, as are antibodies that bind one or more of thesepolypeptides. Moreover, fragments and variants of these polypeptides(e.g. fragments as described herein, polypeptides at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides andpolypeptides encoded by the polynucleotide which hybridizes, understringent conditions, to the polynucleotide encoding these polypeptides,or the complement thereof) are encompassed by the invention. Antibodiesthat bind these fragments and variants of the invention are alsoencompassed by the invention. Polynucleotides encoding these fragmentsand variants are also encompassed by the invention.

[0061] Preferred polypeptides of the present invention comprise, oralternatively consist of, one, two, three, four, five, or all five ofthe immunogenic epitopes shown in SEQ ID NO: 20 as residues: Glu-51 toAsn-60, Arg-82 to Gly-152, Ala-156 to Gly-170, Arg-181 to Glu-186, andGlu-294 to Ser-304. Polynucleotides encoding these polypeptides are alsoencompassed by the invention, as are antibodies that bind one or more ofthese polypeptides. Moreover, fragments and variants of thesepolypeptides (e.g. fragments as described herein, polypeptides at least80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to thesepolypeptides and polypeptides encoded by the polynucleotide whichhybridizes, under stringent conditions, to the polynucleotide encodingthese polypeptides, or the complement thereof) are encompassed by theinvention. Antibodies that bind these fragments and variants of theinvention are also encompassed by the invention. Polynucleotidesencoding these fragments and variants are also encompassed by theinvention.

[0062] It has been discovered that this gene is expressed primarily inlarynx tissue, and cancerous tissue thereof, as well as in macrophage.Diseases associated with increased cell survival, or the inhibition ofapoptosis, include cancers (such as follicular lymphomas, carcinomaswith p53 mutations, and hormone-dependent tumors, including, but notlimited to colon cancer, cardiac tumors, pancreatic cancer, melanoma,retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicularcancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma,endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposiíssarcoma and ovarian cancer); autoimmune disorders (such as, multiplesclerosis, Sjogrenís syndrome, Hashimotoís thyroiditis, biliarycirrhosis, Behcetís disease, Crohnís disease, polymyositis, systemiclupus erythematosus and immune-related glomerulonephritis and rheumatoidarthritis) and viral infections (such as herpes viruses, pox viruses andadenoviruses), inflammation, graft v. host disease, acute graftrejection, and chronic graft rejection. In preferred embodiments, TNFRpolynucleotides, polypeptides, and/or antagonists of the invention areused to inhibit growth, progression, and/or metasis fo cancers, inparticular those listed above. Additional diseases or conditionsassociated with increased cell survival include, but are not limited to,progression, and/or metastases of malignancies and related disorderssuch as leukemia (including acute leukemias (e.g., acute lymphocyticleukemia, acute myelocytic leukemia (including myeloblastic,promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) andchronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia andchronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g.,Hodgkin's disease and non-Hodgkin's disease), multiple myeloma,Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumorsincluding, but not limited to, sarcomas and carcinomas such asfibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma. Diseases associated with increased apoptosis includeAIDS; neurodegenerative disorders (such as Alzheimer's disease,Parkinson's disease, Amyotrophic lateral sclerosis, Retinitispigmentosa, Cerebellar degeneration and brain tumor or prior associateddisease); autoimmune disorders (such as, multiple sclerosis, Sjogreníssyndrome, Hashimotoís thyroiditis, biliary cirrhosis, Behcetís disease,Crohnís disease, polymyositis, systemic lupus erythematosus andimmune-related glomerulonephritis and rheumatoid arthritis)myelodysplastic syndromes (such as aplastic anemia), graft v. hostdisease, ischemic injury (such as that caused by myocardial infarction,stroke and reperfusion injury), liver injury (e.g., hepatitis relatedliver injury, ischemia/reperfusion injury, cholestosis (bile ductinjury) and liver cancer); toxin-induced liver disease (such as thatcaused by alcohol), septic shock, cachexia and anorexia.

[0063] Therefore, polynucleotides and polypeptides of the invention,including antibodies, are useful as reagents for differentialidentification of the tissue(s) or cell type(s) present in a biologicalsample and for diagnosis of the following diseases and conditions:diseases involving aberrant apoptosis, cancers, immune system disorders,or pulmonary disorders. Similarly, polypeptides and antibodies directedto those polypeptides are useful to provide immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of theimmune and pulmonary systems, expression of this gene at significantlyhigher or lower levels may be detected in certain tissues (e.g., immune,pulmonary, cancerous and wounded tissues) or bodily fluids (e.g., lymph,serum, plasma, urine, synovial fluid or spinal fluid) taken from anindividual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue from anindividual not having the disorder.

[0064] The tissue distribution and homology to DEDD suggests that theprotein product of this clone is useful for the diagnosis and/ortreatment of diseases involving aberrant apoptosis. Furthermore, thehomology of the translation product of this gene to DEDD implicates thisgene as being involved in the apoptotic pathway. Therefore, this genemay be important in controlling cell death, and thus, could be extremelyimportant in controlling tumor proliferation.

[0065] Furthermore, the tissue distribution in larynx carcinoma tissuesupports the notion that this gene may somehow be involved in aberrantcell proliferation, or aberrant apoptosis. Similarly, expression of thisgene product in macrophage also strongly suggests a role for thisprotein in immune function and immune surveillance. Protein, as well as,antibodies directed against the protein may show utility as a tumormarker and/or immunotherapy targets for the above listed tissues.

[0066] The ability of polynucleotides and polypeptides of the inventionto increase or decrease apoptosis can routinely be determined usingtechniques known in the art. For example, biological activity canroutinely be measured using cell death assays performed essentially aspreviously described (Chinnaiyan et al., Cell 81:505-512 (1995); Boldinet al., J. Biol. Chem. 270:7795-8(1995); Kischkel et al., EMBO14:5579-5588 (1995); Chinnaiyan et al., J. Biol. Chem. 271:4961-4965(1996)).

[0067] Features of Protein Encoding by Gene No: 2

[0068] For the purposes of the present application, this gene andtranslation products corresponding to this gene may be referred to asNod1. Nod1 polypeptides are thought to function as enhancers ofCaspase-9 activity and NF-kB activation (Examples 17 and 18).Translation products corresponding to this gene share sequence homologywith inhibitor of apoptosis protein 2, which is a member of the humanfamily of inhibitor of apoptosis genes, which are thought to function asapoptotic suppressors (See Genbank Accession AAC50372, and Nature Jan.25, 1996;379(6563):349-53).

[0069] In specific embodiments, polypeptides of the invention comprise,or alternatively consist of, the following amino acid sequence:TAWPASWTTPPASS (SEQ ID NO: 21). Polynucleotides encoding thesepolypeptides are also encompassed by the invention, as are antibodiesthat bind one or more of these polypeptides. Moreover, fragments andvariants of these polypeptides (e.g. fragments as described herein,polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to these polypeptides and polypeptides encoded by thepolynucleotide which hybridizes, under stringent conditions, to thepolynucleotide encoding these polypeptides, or the complement thereof)are encompassed by the invention. Antibodies that bind these fragmentsand variants of the invention are also encompassed by the invention.Polynucleotides encoding these fragments and variants are alsoencompassed by the invention.

[0070] In further specific embodiments of the invention, polypeptides ofthe invention comprise, or alternatively consist of, the following aminoacid sequence: MSRDLLFKHYCYPERDPEEVFAFLLRFPHVALFTFDGLDELHSDLDLSRVPDSSCPWEPAHPLVLLANLLSGKLLKGASKLLTARTGIEVPRQFLRKKVLLRGFSPSHLRAYARRMFPERALQDRLLSQLEANPNLCSLCSVPLFCWIIFRCFQHFRAAFEGSPQLPDCTMTLTDVFLLVTEVHLNRMQPSSLVQRNTRSPVETLHAGRDTLCSLGQVAEHRGMEKSLFVFTQEEVXASGLQERDMQLGFLRALPELGPGGDQQXYEFFHLTLQAFFTAFFLVLDDRVGTQELLRFFQEWMPPAGAATTSCYPPFLPFQCLQGSGPAREDLFKNKDHFQFTNLFLCGLLSKAKQKLLRHLVPAAALRRKRKALWAHLFSSLRGYLKSLPRVQVESFNQVQAMPTFTWMLRCIYETQSQKVGQLAARGICANYLKLTYCNACSADCSALSFVLHHFPKRLALDLDNNNLNDYGVRELQPCFSRLTVLRLSVNQITDGGVKVLSEELTKYKIVTYLGLYNNQITDVGARYVTKILDECKGLTHLKLGKNKITSEGGKYLALAVKNSKSISEVGMWGNQVGDEGAKAFAEALRNHPSLTTLSLASNGISTEGGKSLARALQQNTSLEILWLTQNELXDEXAESLAEMLKVNQTLKHLWLIQNQITAKGTAQLADALQSNTGITEICLNGNLIKPEEAKVYEDE KRIICF (SEQ IDNO:22).

[0071] Polynucleotides encoding these polypeptides are also encompassedby the invention, as are antibodies that bind one or more of thesepolypeptides. Moreover, fragments and variants of these polypeptides(e.g. fragments as described herein, polypeptides at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides andpolypeptides encoded by the polynucleotide which hybridizes, understringent conditions, to the polynucleotide encoding these polypeptides,or the complement thereof) are encompassed by the invention. Antibodiesthat bind these fragments and variants of the invention are alsoencompassed by the invention. Polynucleotides encoding these fragmentsand variants are also encompassed by the invention.

[0072] Preferred polypeptides of the present invention comprise, oralternatively consist of, one, two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, or all fourteen ofthe immunogenic epitopes shown in SEQ ID NO: 21 as residues: Met-1 toSer-7, Pro-62 to Val-68, Ser-77 to Val-82, Pro-125 to Gln-132, Pro-261to Glu-266, Pro-299 to Trp-305, Val-442 to Pro-449, His-454 to Thr-459,Pro-506 to Tyr-513, Asp-577 to Asp-583, Tyr-660 to Lys-666, Leu-710 toTyr-718, Gly-792 to Lys-802, and Thr-876 to Asn-881. Polynucleotidesencoding these polypeptides are also encompassed by the invention, asare antibodies that bind one or more of these polypeptides. Moreover,fragments and variants of these polypeptides (e.g. fragments asdescribed herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to these polypeptides and polypeptides encoded bythe polynucleotide which hybridizes, under stringent conditions, to thepolynucleotide encoding these polypeptides, or the complement thereof)are encompassed by the invention. Antibodies that bind these fragmentsand variants of the invention are also encompassed by the invention.Polynucleotides encoding these fragments and variants are alsoencompassed by the invention.

[0073] It has been discovered that this gene is expressed primarily in awide variety of cancerous and tumor tissues, such as colon cancertissue, thyroid tumor tissue, pancreatic tumor tissue, testes tumortissue, and endometrial tumor tissue. Diseases associated with increasedcell survival, or the inhibition of apoptosis, include cancers (such asfollicular lymphomas, carcinomas with p53 mutations, andhormone-dependent tumors, including, but not limited to colon cancer,cardiac tumors, pancreatic cancer, melanoma, retinoblastoma,glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomachcancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma,osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma,breast cancer, prostate cancer, Kaposiís sarcoma and ovarian cancer);autoinimune disorders (such as, multiple sclerosis, Sjogrenís syndrome,Hashimotoís thyroiditis, biliary cirrhosis, Behcetís disease, Crohnísdisease, polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis and rheumatoid arthritis) and viral infections (suchas herpes viruses, pox viruses and adenoviruses), inflammation, graft v.host disease, acute graft rejection, and chronic graft rejection. Inpreferred embodiments, TNFR polynucleotides, polypeptides, and/orantagonists of the invention are used to inhibit growth, progression,and/or metasis fo cancers, in particular those listed above. Additionaldiseases or conditions associated with increased cell survival include,but are not limited to, progression, and/or metastases of malignanciesand related disorders such as leukemia (including acute leukemias (e.g.,acute lymphocytic leukemia, acute myelocytic leukemia (includingmyeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, and retinoblastoma. Diseases associated withincreased apoptosis include AIDS; neurodegenerative disorders (such asAlzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis,Retinitis pigmentosa, Cerebellar degeneration and brain tumor or priorassociated disease); autoimmune disorders (such as, multiple sclerosis,Sjogrenís syndrome, Hashimotoís thyroiditis, biliary cirrhosis, Behcetísdisease, Crohnís disease, polymyositis, systemic lupus erythematosus andimmune-related glomerulonephritis and rheumatoid arthritis)myelodysplastic syndromes (such as aplastic anemia), graft v. hostdisease, ischemic injury (such as that caused by myocardial infarction,stroke and reperfusion injury), liver injury (e.g., hepatitis relatedliver injury, ischemia/reperfusion injury, cholestosis (bile ductinjury) and liver cancer); toxin-induced liver disease (such as thatcaused by alcohol), septic shock, cachexia and anorexia.

[0074] Therefore, polynucleotides and polypeptides of the invention,including antibodies, are useful as reagents for differentialidentification of the tissue(s) or cell type(s) present in a biologicalsample and for diagnosis of the following diseases and conditions:disorders involving aberrant apoptosis, and cancer. Similarly,polypeptides and antibodies directed to those polypeptides are useful toprovide immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of tumor tissues, expression of this geneat significantly higher or lower levels may be detected in certaintissues (e.g., reproductive, endocrine, gastrointestinal, cancerous andwounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine,synovial fluid or spinal fluid) taken from an individual having such adisorder, relative to the standard gene expression level, i.e., theexpression level in healthy tissue from an individual not having thedisorder.

[0075] Translation products corresponding to this gene may be involvedin pro-apoptotic activities such as the activation of Caspases, such asCaspase-9 (Examples 17 and 18). Accordingly, Nod1 polynucleotides andpolypeptides, or agonists and/or antagonists of Nod1 polynucleotides andpolypeptides, may be useful for the promotion and/or inhibition,respectively, of apoptotic activities mediated by Nod1 polynucleotidesand polypeptides, particularly polypeptide fragments such as CARDdomains.

[0076] In addition, the tissue distribution in a wide variety of tumortissues, and the homology to inhibitor of apoptosis protein 2 (a memberof the inhibitor of apoptosis family of genes), suggests that theprotein product of this clone is useful for the detection and/ortreatment of disorders involving aberrant apoptosis, such as cancer.Furthermore, the homology of the translation product of this gene toinhibitor of apoptosis protein 2 potentially implicates this gene in theregulation of the apoptotic pathway, and therefore, this gene and itstranslation product are potentially useful in controlling aberrant cellgrowth and/or apoptosis, as one might find in cancerous tissues and/ormalignant cells. Protein, as well as, antibodies directed against theprotein may show utility as a tumor marker and/or immunotherapy targetsfor the above listed tissues.

[0077] Alternatively, in a further aspect, the present invention isdirected to a method for inhibiting apoptosis induced by a TNF-familyligand, which involves administering to a cell which expresses thehomolog of the inhibitor of apoptosis protein 2 polypeptide of thepresent invention, an analog or an agonist capable of increasingapoptotic suppression signaling mediated by the homolog of the inhibitorof apoptosis protein 2 polypeptide of the present invention. Preferably,apoptotic signaling is decreased to treat a disease wherein increasedapoptosis, NFkB expression and/or JNK expression is exhibited.

[0078] The ability of polynucleotides and polypeptides of the inventionto increase or decrease apoptosis can routinely be determined usingtechniques known in the art. For example, biological activity canroutinely be measured using cell death assays performed essentially aspreviously described (Chinnaiyan et al., Cell 81:505-512 (1995); Boldinet al., J. Biol. Chem. 270:7795-8(1995); Kischkel et al., EMBO14:5579-5588 (1995); Chinnaiyan et al., J. Biol. Chem. 271:4961-4965(1996)).

[0079] Features of Protein Endoding by Gene No: 3

[0080] Translation products corresponding to this gene share sequencehomology with murine Fas-associated factor 1 (FAF1), which is a proteinwhich specifically interacts with the cytoplasmic domain of wild-typeFas in mice. Thus, it is thought that FAF1 may potentiate Fas-inducedcell killing, and therefore be a candidate signal transducing moleculein the regulation of apoptosis (See Genbank Accession AAA92091, as wellas Proc Natl Acad Sci U S A Dec. 5, 1995;92(25):11894-8). Additionally,the translation product of this gene shares sequence homology with theDrosophila Fly Fas-associated factor (FFAF), which is a homolog of themurine FAF1 (See Genbank Accession BAA33466).

[0081] Preferred polypeptides of the present invention comprise, oralternatively consist of, one, two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, or all fourteen ofthe immunogenic epitopes shown in SEQ ID NO: 22 as residues: Ala-3 toLys-16, Arg-50 to Gly-56, Arg-85 to Gly-90, Pro-123 to Val-129, Glu-144to Arg-149, Asn-164 to Glu-169, His-178 to Cys-189, Ser-212 to Tyr-219,Arg-277 to Glu-295, Ala-303 to Lys-346, Pro-352 to Glu-360, Pro-369 toHis-379, Lys-395 to Lys-400, Pro-416 to Leu-426. Polynucleotidesencoding these polypeptides are also encompassed by the invention, asare antibodies that bind one or more of these polypeptides. Moreover,fragments and variants of these polypeptides (e.g. fragments asdescribed herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to these polypeptides and polypeptides encoded bythe polynucleotide which hybridizes, under stringent conditions, to thepolynucleotide encoding these polypeptides, or the complement thereof)are encompassed by the invention. Antibodies that bind these fragmentsand variants of the invention are also encompassed by the invention.Polynucleotides encoding these fragments and variants are alsoencompassed by the invention.

[0082] It has been discovered that this gene is expressed primarily inimmune cells such as monocytes, primary dendritic cells, fetalliver/spleen tissue, and bone marrow, and to a lesser extent in vasculartissues such as heart tissue. Diseases associated with increased cellsurvival, or the inhibition of apoptosis, include cancers (such asfollicular lymphomas, carcinomas with p53 mutations, andhormone-dependent tumors, including, but not limited to colon cancer,cardiac tumors, pancreatic cancer, melanoma, retinoblastoma,glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomachcancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma,osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma,breast cancer, prostate cancer, Kaposiís sarcoma and ovarian cancer);autoimmune disorders (such as, multiple sclerosis, Sjogrenís syndrome,Hashimotoís thyroiditis, biliary cirrhosis, Behcetís disease, Crohnísdisease, polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis and rheumatoid arthritis) and viral infections (suchas herpes viruses, pox viruses and adenoviruses), inflammation, graft v.host disease, acute graft rejection, and chronic graft rejection. Inpreferred embodiments, TNFR polynucleotides, polypeptides, and/orantagonists of the invention are used to inhibit growth, progression,and/or metasis fo cancers, in particular those listed above. Additionaldiseases or conditions associated with increased cell survival include,but are not limited to, progression, and/or metastases of malignanciesand related disorders such as leukemia (including acute leukemias (e.g.,acute lymphocytic leukemia, acute myelocytic leukemia (includingmyeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, and retinoblastoma. Diseases associated withincreased apoptosis include AIDS; neurodegenerative disorders (such asAlzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis,Retinitis pigmentosa, Cerebellar degeneration and brain tumor or priorassociated disease); autoimmune disorders (such as, multiple sclerosis,Sjogrenís syndrome, Hashimotoís thyroiditis, biliary cirrhosis, Behcetísdisease, Crohnís disease, polymyositis, systemic lupus erythematosus andimmune-related glomerulonephritis and rheumatoid arthritis)myelodysplastic syndromes (such as aplastic anemia), graft v. hostdisease, ischemic injury (such as that caused by myocardial infarction,stroke and reperfusion injury), liver injury (e.g., hepatitis relatedliver injury, ischemia/reperfusion injury, cholestosis (bile ductinjury) and liver cancer); toxin-induced liver disease (such as thatcaused by alcohol), septic shock, cachexia and anorexia.

[0083] Therefore, polynucleotides and polypeptides of the invention,including antibodies, are useful as reagents for differentialidentification of the tissue(s) or cell type(s) present in a biologicalsample and for diagnosis of the following diseases and conditions:immune system disorders, and aberrant apoptosis. Similarly, polypeptidesand antibodies directed to those polypeptides are useful to provideimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the immune system, expression of this gene atsignificantly higher or lower levels may be detected in certain tissues(e.g., immune, vascular, cancerous and wounded tissues) or bodily fluids(e.g., lymph, serum, plasma, urine, synovial fluid or spinal fluid)taken from an individual having such a disorder, relative to thestandard gene expression level, i.e., the expression level in healthytissue from an individual not having the disorder.

[0084] The tissue distribution in immune tissues, and the homology tothe murine FAF1, as well as the Drosophila FFAF, suggests that theprotein product of this clone is useful for the detection and/ortreatment of disorders involving aberrant apoptosis. The homology of thetranslation product of this gene to both FAF1 and FFAF strongly suggeststhat this gene is involved in the regulation of the apoptotic pathway,most likely as a protein that directly interacts with Fas, and therebymediates Fas-associated apoptosis and cell death. Therefore, this genewould be useful for the detection and/or treatment of disordersinvolving aberrant cell growth and or apoptosis, such as one might findin cancerous tissues and/or malignant cells. Protein, as well as,antibodies directed against the protein may show utility as a tumormarker and/or immunotherapy targets for the above listed tissues.

[0085] Thus, in one aspect, the present invention is directed to amethod for enhancing apoptosis induced by a TNF-family ligand, whichinvolves administering to a cell which expresses the polypeptide of theFAF1 homolog of the present invention, or an agonist capable ofincreasing signaling mediated by the polypeptide of the FAF1 homolog ofthe present invention. Preferably, apoptotic signalling mediated by thepolypeptide of the FAF1 homolog of the present invention is increased totreat a disease wherein decreased apoptosis or decreased cytokine andadhesion molecule expression is exhibited. Agonists include, but are notlimited to, soluble forms of the polypeptide of the FAF1 homolog of thepresent invention and antibodies (preferably monoclonal) directedagainst the polypeptide of the FAF1 homolog of the present invention.

[0086] The ability of polynucleotides and polypeptides of the inventionto increase or decrease apoptosis can routinely be determined usingtechniques known in the art. For example, biological activity canroutinely be measured using cell death assays performed essentially aspreviously described (Chinnaiyan et al., Cell 81:505-512 (1995); Boldinet al., J. Biol. Chem. 270:7795-8(1995); Kischkel et al., EMBO14:5579-5588 (1995); Chinnaiyan et al., J. Biol. Chem. 271:4961-4965(1996)).

[0087] Features of Protein Encoded by Gene No: 4

[0088] Translation products corresponding to this gene share sequencehomology with human receptor interacting protein (RIP). When RIP istransiently overexpressed, transfected cells undergo the morphologicalchanges characteristic of apoptosis. Thus, RIP is thought to be a novelform of an apoptosis-inducing protein (See Genbank Accession AAC50137,as well as Cell May 19, 1995; 81(4):513-23). In addition to RIP, as wellas FADD and TRADD, there are other non-receptor proteins that containdeath domains and participate in transducing signals. Some serve asadaptor molecules to bring various components to the signaling complex.RAIDD, for example, which causes apoptosis when overexpressed, binds tothe homologous domain of RIP through the death domain and to caspase-2through another part of the molecule. The binding results suggested thatRAIDD functions as an adaptor to recruit caspase-2 to the TNFR1signaling complex. RAIDD, however, may not be essential for apoptosis,since various putative dominant mutants of RAIDD failed to blockTNFR1-induced apoptosis. MADD associates with the death domain of TNFR1and activates mitogen-activated kinase, another signal transducedthrough the TNFR1. An interesting example of a death domain in anadaptor protein is Siva, which forms a complex with CD27, a non-deathdomain-containing receptor, and induces apoptosis. Deathdomain-containing intracellular proteins have also been found in otherorganisms, for example, reaper, which is in Drosophila.

[0089] In specific embodiments, polypeptides of the invention comprise,or alternatively consist of, the following amino acid sequence:APCCACHRAVPPASSNRSPCSCLCPLASQASVWTAPACTCCTGPLLQPPG (SEQ ID NO: 23).Polynucleotides encoding these polypeptides are also encompassed by theinvention, as are antibodies that bind one or more of thesepolypeptides. Moreover, fragments and variants of these polypeptides(e.g. fragments as described herein, polypeptides at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides andpolypeptides encoded by the polynucleotide which hybridizes, understringent conditions, to the polynucleotide encoding these polypeptides,or the complement thereof) are encompassed by the invention. Antibodiesthat bind these fragments and variants of the invention are alsoencompassed by the invention. Polynucleotides encoding these fragmentsand variants are also encompassed by the invention.

[0090] Preferred polypeptides of the present invention comprise, oralternatively consist of, one, two, three, four, or all four of theimmunogenic epitopes shown in SEQ ID NO: 23 as residues: Arg-53 toGlu-58, Glu-79 to Asp-88, Asp-109 to Val-116, and Leu-190 to Ala-200.Polynucleotides encoding these polypeptides are also encompassed by theinvention, as are antibodies that bind one or more of thesepolypeptides. Moreover, fragments and variants of these polypeptides(e.g. fragments as described herein, polypeptides at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides andpolypeptides encoded by the polynucleotide which hybridizes, understringent conditions, to the polynucleotide encoding these polypeptides,or the complement thereof) are encompassed by the invention. Antibodiesthat bind these fragments and variants of the invention are alsoencompassed by the invention. Polynucleotides encoding these fragmentsand variants are also encompassed by the invention.

[0091] It has been discovered that this gene is expressed primarily inimmune system cells and tissues such as monocytes and fetal liver/spleentissue, and to a lesser extent in Wilm's tumor and breast cancertissues. Diseases associated with increased cell survival, or theinhibition of apoptosis, include cancers (such as follicular lymphomas,carcinomas with p53 mutations, and hormone-dependent tumors, including,but not limited to colon cancer, cardiac tumors, pancreatic cancer,melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer,testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma,lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposiíssarcoma and ovarian cancer); autoimmune disorders (such as, multiplesclerosis, Sjogrenís syndrome, Hashimotoís thyroiditis, biliarycirrhosis, Behcetís disease, Crohnís disease, polymyositis, systemiclupus erythematosus and immune-related glomerulonephritis and rheumatoidarthritis) and viral infections (such as herpes viruses, pox viruses andadenoviruses), inflammation, graft v. host disease, acute graftrejection, and chronic graft rejection. In preferred embodiments, TNFRpolynucleotides, polypeptides, and/or antagonists of the invention areused to inhibit growth, progression, and/or metasis fo cancers, inparticular those listed above. Additional diseases or conditionsassociated with increased cell survival include, but are not limited to,progression, and/or metastases of malignancies and related disorderssuch as leukemia (including acute leukemias (e.g., acute lymphocyticleukemia, acute myelocytic leukemia (including myeloblastic,promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) andchronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia andchronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g.,Hodgkin's disease and non-Hodgkin's disease), multiple myeloma,Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumorsincluding, but not limited to, sarcomas and carcinomas such asfibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma. Diseases associated with increased apoptosis includeAIDS; neurodegenerative disorders (such as Alzheimer's disease,Parkinson's disease, Amyotrophic lateral sclerosis, Retinitispigmentosa, Cerebellar degeneration and brain tumor or prior associateddisease); autoimmune disorders (such as, multiple sclerosis, Sjogreníssyndrome, Hashimotoís thyroiditis, biliary cirrhosis, Behcetís disease,Crohnís disease, polymyositis, systemic lupus erythematosus andimmune-related glomerulonephritis and rheumatoid arthritis)myelodysplastic syndromes (such as aplastic anemia), graft v. hostdisease, ischemic injury (such as that caused by myocardial infarction,stroke and reperfusion injury), liver injury (e.g., hepatitis relatedliver injury, ischemia/reperfusion injury, cholestosis (bile ductinjury) and liver cancer); toxin-induced liver disease (such as thatcaused by alcohol), septic shock, cachexia and anorexia.

[0092] Therefore, polynucleotides and polypeptides of the invention,including antibodies, are useful as reagents for differentialidentification of the tissue(s) or cell type(s) present in a biologicalsample and for diagnosis of the following diseases and conditions:disorders involving aberrant apoptosis, and immune system disorders.Similarly, polypeptides and antibodies directed to those polypeptidesare useful to provide immunological probes for differentialidentification of the tissue(s) or cell type(s). For a number ofdisorders of the above tissues or cells, particularly of the immunesystem, expression of this gene at significantly higher or lower levelsmay be detected in certain tissues (e.g., immune, cancerous and woundedtissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovialfluid or spinal fluid) taken from an individual having such a disorder,relative to the standard gene expression level, i.e., the expressionlevel in healthy tissue from an individual not having the disorder.

[0093] The tissue distribution in immune system tissues and cells, andthe homology to human RIP, suggests that the protein product of thisclone is useful for the diagnosis and/or treatment of disordersinvolving aberrant apoptosis. Furthermore, the homology of thetranslation product of this gene to RIP strongly suggests that thetranslation product of this gene may be involved in the regulation ofthe apoptotic pathway, in particular in the promotion of apoptosis.Thus, the gene, and its corresponding translation product, are usefulfor the diagnosis and/or treatment of disorders involving aberrantapoptosis, such as one might find in cancerous tissues and/or malignantcells. Protein, as well as, antibodies directed against the protein mayshow utility as a tumor marker and/or immunotherapy targets for theabove listed tissues.

[0094] Thus, in one aspect, the present invention is directed to amethod for enhancing apoptosis induced by a TNF-family ligand, whichinvolves administering to a cell which expresses the polypeptide of theRIP homolog of the present invention, or an agonist capable ofincreasing signaling mediated by the polypeptide of the RIP homolog ofthe present invention. Preferably, apoptotic signaling mediated by thepolypeptide of the RIP homolog of the present invention is increased totreat a disease wherein decreased apoptosis or decreased cytokine andadhesion molecule expression is exhibited. Agonists include, but are notlimited to, soluble forms of the polypeptide of the RIP homolog of thepresent invention and antibodies (preferably monoclonal) directedagainst the polypeptide of the RIP homolog of the present invention.

[0095] The ability of polynucleotides and polypeptides of the inventionto increase or decrease apoptosis can routinely be determined usingtechniques known in the art. For example, biological activity canroutinely be measured using cell death assays performed essentially aspreviously described (Chinnaiyan et al., Cell 81:505-512 (1995); Boldinet al., J. Biol. Chem. 270:7795-8(1995); Kischkel et al., EMBO14:5579-5588 (1995); Chinnaiyan et al., J. Biol. Chem. 271:4961-4965(1996)).

[0096] Features of Protein Encoding by Gene No: 5

[0097] Translation products corresponding to this gene share sequencehomology with human cystein rich domain associated to RING and TRAFprotein (See Genbank Accession CAA56491), which is thought to beinvolved in the regulation of the apoptotic pathway. A group ofproteins, whose first member was isolated based on its association withTNFR2, also function as adaptors for transducing signals. They aredefined by the presence of a C-terminal domain, designated TRAF, ofabout 230 amino acids. This region has been further divided into TRAF-Nand TRAF-C subdomains. Several of the 6 TRAFs identified thus farinteract with many receptors belonging to the TNFR superfamily, butthere is only limited direct association of any of the TRAFs with deathdomain-containing receptors. TRAF2 is recruited to the TNFR1 signalingcomplex through TRADD. Similarly, there is only a weak association ofTRAF2 with DR3, but the association is enhanced when TRADD isoverexpressed. TRAF1 and TRAF2 are involved in CD30-mediated cell deathof T cell hybridomas, and overexpression of TRAF1 in transgenic miceinhibits antigen-induced apoptosis of CD8+ lymphocytes.

[0098] Preferred polypeptides of the present invention comprise, oralternatively consist of, one, two, three, four, five, six, or all sixof the immunogenic epitopes shown in SEQ ID NO: 24 as residues: Phe-47to Thr-52, Ser-104 to Thr-115, Pro-125 to Asn-131, Gln-143 to Arg-148,Glu-153 to Glu-158, and Pro-185 to Leu-190. Polynucleotides encodingthese polypeptides are also encompassed by the invention, as areantibodies that bind one or more of these polypeptides. Moreover,fragments and variants of these polypeptides (e.g. fragments asdescribed herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to these polypeptides and polypeptides encoded bythe polynucleotide which hybridizes, under stringent conditions, to thepolynucleotide encoding these polypeptides, or the complement thereof)are encompassed by the invention. Antibodies that bind these fragmentsand variants of the invention are also encompassed by the invention.Polynucleotides encoding these fragments and variants are alsoencompassed by the invention.

[0099] It has been discovered that this gene is expressed primarily inneural tissues such as infant brain, and to a lesser extent inmusculo-skeletal tissues such as muscule tissue and synovial hypoxiatissues.

[0100] Therefore, polynucleotides and polypeptides of the invention,including antibodies, are useful as reagents for differentialidentification of the tissue(s) or cell type(s) present in a biologicalsample and for diagnosis of the following diseases and conditions:disorders associated with aberrant apoptosis, neural disorders, andmusculo-skeletal disorders. Similarly, polypeptides and antibodiesdirected to those polypeptides are useful to provide immunologicalprobes for differential identification of the tissue(s) or cell type(s).For a number of disorders of the above tissues or cells, particularly ofthe neural and musculo-skeletal systems, expression of this gene atsignificantly higher or lower levels may be detected in certain tissues(e.g., neural, musculo-skeletal, cancerous and wounded tissues) orbodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid orspinal fluid) taken from an individual having such a disorder, relativeto the standard gene expression level, i.e., the expression level inhealthy tissue from an individual not having the disorder.

[0101] The homology of the translation product of this clone to humancystein rich domain associated to RING and TRAF protein, suggests thatthe protein product of this clone is useful for the diagnosis and/ortreatment of disorders involving aberrant apoptosis. Furthermore, giventhe homology of the translation product of this clone, it is possiblethat this gene is involved in the apoptotic pathway, and thus, could beuseful in controlling aberrant cell growth or apoptosis, as one mightfind in cancerous tissues and/or malignant cells.

[0102] Alternatively, given the tissue distribution in neural tissuessuch as infant brain, the protein product of this clone may be usefulfor the detection/treatment of neurodegenerative disease states andbehavioural disorders such as Alzheimers Disease, Parkinsons Disease,Huntingtons Disease, Tourette Syndrome, schizophrenia, mania, dementia,paranoia, obsessive compulsive disorder, panic disorder, learningdisabilities, ALS, psychoses, autism, and altered behaviors, includingdisorders in feeding, sleep patterns, balance, and perception. Inaddition, the gene or gene product may also play a role in the treatmentand/or detection of developmental disorders associated with thedeveloping embryo, or sexually-linked disorders.

[0103] Likewise, the tissue distribution in muscle tissue and indicatesthat the protein product of this gene is useful for the diagnosis andtreatment of conditions and pathologies of the cardiovascular system,such as heart disease, restenosis, atherosclerosis, stoke, angina,thrombosis, and wound healing. In addition, the expression of this geneproduct in synovium suggests a role in the detection and treatment ofdisorders and conditions affecting the skeletal system, in particularosteoporosis as well as disorders afflicting connective tissues (e.g.arthritis, trauma, tendonitis, chrondomalacia and inflammation), such asin the diagnosis or treatment of various autoimmune disorders such asrheumatoid arthritis, lupus, scleroderma, and dermatomyositis as well asdwarfism, spinal deformation, and specific joint abnormalities as wellas chondrodysplasias (ie. spondyloepiphyseal dysplasia congenita,familial arthritis, Atelosteogenesis type II, metaphysealchondrodysplasia type Schmid). Protein, as well as, antibodies directedagainst the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0104] The ability of polynucleotides and polypeptides of the inventionto increase or decrease apoptosis can routinely be determined usingtechniques known in the art. For example, biological activity canroutinely be measured using cell death assays performed essentially aspreviously described (Chinnaiyan et al., Cell 81:505-512 (1995); Boldinet al., J. Biol. Chem. 270:7795-8(1995); Kischkel et al., EMBO14:5579-5588 (1995); Chinnaiyan et al., J. Biol. Chem. 271:4961-4965(1996)).

[0105] Features of Protein Encoding by Gene No: 6

[0106] Translation products corresponding to this gene share sequencehomology with human eosinophil peroxidase, which is thought to beimportant in immune system function and surveillance (See GenbankAccession AAA58458), as well as with hSNF2b (See Genbank AccessionBAA05143), which is thought to function as a transcriptional activator.

[0107] In specific embodiments, polypeptides of the invention comprise,or alternatively consist of, the following amino acid sequence:AWWRRKGTWPWTCSSEALVKGTLTSCPILDSICK (SEQ ID NO: 24). Polynucleotidesencoding these polypeptides are also encompassed by the invention, asare antibodies that bind one or more of these polypeptides. Moreover,fragments and variants of these polypeptides (e.g. fragments asdescribed herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to these polypeptides and polypeptides encoded bythe polynucleotide which hybridizes, under stringent conditions, to thepolynucleotide encoding these polypeptides, or the complement thereof)are encompassed by the invention. Antibodies that bind these fragmentsand variants of the invention are also encompassed by the invention.Polynucleotides encoding these fragments and variants are alsoencompassed by the invention.

[0108] Preferred polypeptides of the present invention comprise, oralternatively consist of, one, two, or both of the immunogenic epitopesshown in SEQ ID NO: 25 as residues: Leu-7 to Trp-18 and Gln-30 toAsn-37. Polynucleotides encoding these polypeptides are also encompassedby the invention, as are antibodies that bind one or more of thesepolypeptides. Moreover, fragments and variants of these polypeptides(e.g. fragments as described herein, polypeptides at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides andpolypeptides encoded by the polynucleotide which hybridizes, understringent conditions, to the polynucleotide encoding these polypeptides,or the complement thereof) are encompassed by the invention. Antibodiesthat bind these fragments and variants of the invention are alsoencompassed by the invention. Polynucleotides encoding these fragmentsand variants are also encompassed by the invention.

[0109] It has been discovered that this gene is expressed primarily inprimary dendritic cells, and to a lesser extent in ovarian tumor tissue.

[0110] Therefore, polynucleotides and polypeptides of the invention,including antibodies, are useful as reagents for differentialidentification of the tissue(s) or cell type(s) present in a biologicalsample and for diagnosis of the following diseases and conditions:immune system disorders, and aberrant apoptosis. Similarly, polypeptidesand antibodies directed to those polypeptides are useful to provideimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the immune system, expression of this gene atsignificantly higher or lower levels may be detected in certain tissues(e.g., immune, cancerous and wounded tissues) or bodily fluids (e.g.,lymph, serum, plasma, urine, synovial fluid or spinal fluid) taken froman individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue from anindividual not having the disorder.

[0111] The tissue distribution in primary dendritic cells, and thehomology to eosinophil peroxidase, suggests that the protein product ofthis clone is useful for the detection and/or treatment of immune systemdisorders associated with cells involved in immune system function andimmune surveillance.

[0112] Alternatively, the tissue distribution in ovarian tumor tissuesuggests that the translation product of this gene may be involved inapoptosis. Expression within cellular sources marked by proliferatingcells suggests that this protein may play a role in the regulation ofcellular division, and may show utility in the diagnosis and treatmentof cancer and other proliferative disorders. Thus, this protein may alsobe involved in apoptosis and could be useful in cancer therapy. Protein,as well as, antibodies directed against the protein may show utility asa tumor marker and/or immunotherapy targets for the above listedtissues.

[0113] The ability of polynucleotides and polypeptides of the inventionto increase or decrease apoptosis can routinely be determined usingtechniques known in the art. For example, biological activity canroutinely be measured using cell death assays performed essentially aspreviously described (Chinnaiyan et al., Cell 81:505-512 (1995); Boldinet al., J. Biol. Chem. 270:7795-8(1995); Kischkel et al., EMBO14:5579-5588 (1995); Chinnaiyan et al., J. Biol. Chem. 271:4961-4965(1996)).

[0114] Features of Protein Encoding by Gene No: 7

[0115] Translation products corresponding to this gene share sequencehomology with a C. elegans protein of unknown function (See GenbankAccession AAB37815).

[0116] In specific embodiments, polypeptides of the invention comprise,or alternatively consist of, the following amino acid sequence:QGRFRAFCWQRDFLQPPG (SEQ ID NO: 25). Polynucleotides encoding thesepolypeptides are also encompassed by the invention, as are antibodiesthat bind one or more of these polypeptides. Moreover, fragments andvariants of these polypeptides (e.g. fragments as described herein,polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to these polypeptides and polypeptides encoded by thepolynucleotide which hybridizes, under stringent conditions, to thepolynucleotide encoding these polypeptides, or the complement thereof)are encompassed by the invention. Antibodies that bind these fragmentsand variants of the invention are also encompassed by the invention.Polynucleotides encoding these fragments and variants are alsoencompassed by the invention.

[0117] Preferred polypeptides of the present invention comprise, oralternatively consist of, one, two, three, four, five, six, seven,eight, or all eight of the immunogenic epitopes shown in SEQ ID NO: 26as residues: Pro-17 to Met-23, Ala-30 to Trp-38, Ile-49 to Trp-54,Lys-68 to Gly-74, Thr-93 to Gly-99, Met-126 to Glu-132, Gly-173 toSer-178, and Lys-205 to Tyr-214. Polynucleotides encoding thesepolypeptides are also encompassed by the invention, as are antibodiesthat bind one or more of these polypeptides. Moreover, fragments andvariants of these polypeptides (e.g. fragments as described herein,polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to these polypeptides and polypeptides encoded by thepolynucleotide which hybridizes, under stringent conditions, to thepolynucleotide encoding these polypeptides, or the complement thereof)are encompassed by the invention. Antibodies that bind these fragmentsand variants of the invention are also encompassed by the invention.Polynucleotides encoding these fragments and variants are alsoencompassed by the invention.

[0118] It has been discovered that this gene is expressed primarily inprimary dendritic cells, and to a lesser extent in ovarian tumor tissue.

[0119] Therefore, polynucleotides and polypeptides of the invention,including antibodies, are useful as reagents for differentialidentification of the tissue(s) or cell type(s) present in a biologicalsample and for diagnosis of the following diseases and conditions:immune system disorders and cancers. Similarly, polypeptides andantibodies directed to those polypeptides are useful to provideimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the immune and reproductive systems, expression of thisgene at significantly higher or lower levels may be detected in certaintissues (e.g., immune, reproductive, cancerous and wounded tissues) orbodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid orspinal fluid) taken from an individual having such a disorder, relativeto the standard gene expression level, i.e., the expression level inhealthy tissue from an individual not having the disorder.

[0120] The tissue distribution in primary dendritic cells suggests thatthe protein product of this clone is useful for the detection and/ortreatment of disorders involving the immune system. This gene productmay be involved in the regulation of cytokine production, antigenpresentation, or other processes that may also suggest a usefulness inthe treatment of cancer (e.g. by boosting immune responses). Since thegene is expressed in cells of lymphoid origin, the gene or protein, aswell as, antibodies directed against the protein may show utility as atumor marker and/or immunotherapy targets for the above listed tissues.Therefore it may be also used as an agent for immunological disordersincluding arthritis, asthma, immune deficiency diseases such as AIDS,leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis,acne, and psoriasis. In addition, this gene product may have commercialutility in the expansion of stem cells and committed progenitors ofvarious blood lineages, and in the differentiation and/or proliferationof various cell types.

[0121] Alternatively, the tissue distribution in ovarian cancer tissuesuggests that the translation product of this gene is useful for thedetection and/or treatment of ovarian cancer, as well as cancers ofother tissues where expression has been observed. Furthermore, it ispossible that this gene is involved in the apoptotic pathway, and thus,could be useful in controlling aberrant cell growth or apoptosis, as onemight find in cancerous tissues and/or malignant cells. Protein, as wellas, antibodies directed against the protein may show utility as a tumormarker and/or immunotherapy targets for the above listed tissues.

[0122] Polynucleotides and polypeptides corresponding to this gene arethought to be involved in apoptosis. The ability of polynucleotides andpolypeptides of the invention to increase or decrease apoptosis canroutinely be determined using techniques known in the art. For example,biological activity can routinely be measured using cell death assaysperformed essentially as previously described (Chinnaiyan et al., Cell81:505-512 (1995); Boldin et al., J. Biol. Chem. 270:7795-8(1995);Kischkel et al., EMBO 14.5579-5588 (1995); Chinnaiyan et al., J. Biol.Chem. 271:4961-4965 (1996)).

[0123] Features of Protein Encoded by Gene No: 8

[0124] Preferred polypeptides of the invention comprise, oralternatively consist of, an amino acid sequence selected from the groupconsisting of: MFRCGGLAAGALKQKLVPLVRTVCVRSPRQR (SEQ ID NO:26),andNRLPGNLFQRWHVPLELQMTRQMASSGASGG KIDNSVLVLIVGLSTVGAGAYAYKTMKEDEKRYNERISGLGLTPEQKQKKAALSASEGEEVP QDKAPSHVPFLLIGGGTAAFAAARSIRARDPGARVLIVSEDPELPYMRPPLSKELWFSDDPN VTKTLRFKQWNGKERSIYFQPPSFYVSAQDLPHIENGGVAVLTGKKVVQLDVRDNMVKLNDG SQITYEKCLIATGGTPRSLSAIDRAGAEVKSRTTLFRKIGDFRSLEKISREVKSITIIGGGF LGSELACALGRKARALGTEVIQLFPEKGNMGKILPEYLSNWTMEKVRREGVKVMPNAIVQSV GVSSGKLLIKLKDGRKVETDHIVAAVGLEPNVELAKTGGLEIDSDFGGFRVNAELQARSNIW VAGDAACFYDIKLGRRRVEHHDHAVVSGRLAGENMTGAAKPYWHQSMFWSDLGPDVGYEAIG LVDSSLPTVGVFAKATAQDNPKSATEQSGTGIRSESETESEASEITIPPSTPAVPQAPVQGE DYGKGVIFYLRDKVVVGIVLWNIFNRMPIARKIIKDGEQHEDLNEVAKLFNTHED RTRGSTHASGLTRRSCVRGKGRRRSRIAVAE (SEQ ID NO:27).

[0125] Polynucleotides encoding these polypeptides are also encompassedby the invention, as are antibodies that bind one or more of thesepolypeptides. Moreover, fragments and variants of these polypeptides(e.g. fragments as described herein, polypeptides at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides andpolypeptides encoded by the polynucieotide which hybridizes, understringent conditions, to the polynucleotide encoding these polypeptides,or the complement thereof) are encompassed by the invention. Antibodiesthat bind these fragments and variants of the invention are alsoencompassed by the invention. Polynucleotides encoding these fragmentsand variants are also encompassed by the invention.

[0126] Preferred polypeptides of the present invention comprise, oralternatively consist of, one, two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, or all twelve of the immunogenicepitopes shown in SEQ ID NO: 27 as residues: Ser-27 to Pro-35, Thr-87 toIle-99, Pro-106 to Lys-112, Glu-119 to Ser-130, Ser-182 to Val-187,Lys-194 to Tyr-204, Lys-295 to Val-300, Lys-384 to Glu-390, Thr-512 toThr-526, Ser-530 to Glu-537, Gln-556 to Lys-562, Lys-593 to Leu-601.Polynucleotides encoding these polypeptides are also encompassed by theinvention, as are antibodies that bind one or more of thesepolypeptides. Moreover, fragments and variants of these polypeptides(e.g. fragments as described herein, polypeptides at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides andpolypeptides encoded by the polynucleotide which hybridizes, understringent conditions, to the polynucleotide encoding these polypeptides,or the complement thereof) are encompassed by the invention. Antibodiesthat bind these fragments and variants of the invention are alsoencompassed by the invention. Polynucleotides encoding these fragmentsand variants are also encompassed by the invention.

[0127] It has been discovered that this gene is expressed primarily inthe following tissues/cDNA libraries: primary dendritic cells, lib 1;and to a lesser extent in soares ovary tumor; human testes; soaresparathyroid tumor; human adult small intestine, re-excision; human adultsmall intestine; human heart cDNA; T-cell helper II; and soares fetalliver spleen.

[0128] Therefore, polynucleotides and polypeptides of the invention,including antibodies, are useful as reagents for differentialidentification of the tissue(s) or cell type(s) present in a biologicalsample and for diagnosis of the following diseases and conditions:immune and hematopoietic diseases and/or disorders, particularlyinfectious and proliferative diseases. Similarly, polypeptides andantibodies directed to those polypeptides are useful to provideimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the immune system, expression of this gene atsignificantly higher or lower levels may be detected in certain tissuesor cell types (e.g., immune, hematopoietic, and cancerous and woundedtissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovialfluid or spinal fluid) taken from an individual having such a disorder,relative to the standard gene expression level, i.e., the expressionlevel in healthy tissue from an individual not having the disorder.

[0129] The tissue distribution in dendritic cells indicates the proteinproduct of this clone is useful for the diagnosis and treatment of avariety of immune system disorders. Representative uses are described inthe “Immune Activity” and “Infectious Disease” sections below. Briefly,the expression of this gene product indicates a role in regulating theproliferation; survival; differentiation; and/or activation ofhematopoietic cell lineages, including blood stem cells. This geneproduct is involved in the regulation of cytokine production, antigenpresentation, or other processes suggesting a usefulness in thetreatment of cancer (e.g., by boosting immune responses). Since the geneis expressed in cells of lymphoid origin, the natural gene product isinvolved in immune functions. Therefore it is also used as an agent forimmunological disorders including arthritis, asthma, immunodeficiencydiseases such as AIDS, leukemia, rheumatoid arthritis, granulomatousdisease, inflammatory bowel disease, sepsis, acne, neutropenia,neutrophilia, psoriasis, hypersensitivities, such as T-cell mediatedcytotoxicity; immune reactions to transplanted organs and tissues, suchas host-versus-graft and graft-versus-host diseases, or autoimmunitydisorders, such as autoimmune infertility, lense tissue injury,demyelination, systemic lupus erythematosis, drug induced hemolyticanemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.Moreover, the protein may represent a secreted factor that influencesthe differentiation or behavior of other blood cells, or that recruitshematopoietic cells to sites of injury. Thus, this gene product isthought to be useful in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types.

[0130] Moreover, the expression within cellular sources marked byproliferating cells indicates this protein may play a role in theregulation of cellular division, and may show utility in the diagnosis,treatment, and/or prevention of developmental diseases and disorders,cancer, and other proliferative conditions. Representative uses aredescribed in the “Hyperproliferative Disorders” and “Regeneration”sections below and elsewhere herein. Briefly, developmental tissues relyon decisions involving cell differentiation and/or apoptosis in patternformation.

[0131] Polynucleotides and polypeptides corresponding to this gene arethought to function in apoptosis. Dysregulation of apoptosis can resultin inappropriate suppression of cell death, as occurs in the developmentof some cancers, or in failure to control the extent of cell death, asis believed to occur in acquired immunodeficiency and certainneurodegenerative disorders, such as spinal muscular atrophy (SMA).Because of potential roles in proliferation and differentiation, thisgene product may have applications in the adult for tissue regenerationand the treatment of cancers. It may also act as a morphogen to controlcell and tissue type specification. Therefore, the polynucleotides andpolypeptides of the present invention are useful in treating, detecting,and/or preventing said disorders and conditions, in addition to othertypes of degenerative conditions. Thus translation productscorresponding to this gene may modulate apoptosis or tissuedifferentiation and would be useful in the detection, treatment, and/orprevention of degenerative or proliferative conditions and diseases. Theprotein is useful in modulating the immune response to aberrantpolypeptides, as may exist in proliferating and cancerous cells andtissues. The protein can also be used to gain new insight into theregulation of cellular growth and proliferation.

[0132] Polypeptides of the present invention have been shown to includea flavin moiety, and share sequence homology to bacterialoxidoreductases. Such flavin moieties are subject to regulation eitherby the reduction/oxidation potential of the intracellular environment orthrough the application of specific suicide inhibitors such asN,N-dimethylpropargyalamine, for example (See Enzyme Structure andMechanism; Fersht, Alan; W. H. Freeman and Company, pp.260-261, (1985),which is hereby incorporated herein by reference). Based upon thepotential for such regulation, antagonists for the present invention arecontemplated and would be useful in inhibiting the specific apoptoticpathways subject to regulation by polypeptides of the present invention.Such inhibition would be useful, though not limited to, inhibitingstress-, disease-, and/or infection-induced apoptotic responses, forexample. Polypeptides of the present invention may have other uses,aside from serving as a downstream effector for apoptosis, which aredescribed above, and elseware herein.

[0133] Apoptosis, or programmed cell death, is a physiologic processessential to the normal development and homeostasis of multicellularorganisms (H. Steller, Science 267, 1445-1449 (1995)). Derangements ofapoptosis contribute to the pathogenesis of several human diseasesincluding cancer, neurodegenerative disorders, and acquired immunedeficiency syndrome (C. B. Thompson, Science 267, 1456-1462 (1995)).Recently, much attention has focused on the signal transduction andbiological function of two cell surface death receptors, Fas/APO-1 andTNFR-1 (J. L. Cleveland, et al., Cell 81, 479-482 (1995); A. Fraser, etal., Cell 85, 781-784 (1996); S. Nagata, et al., Science 267, 1449-56(1995)). Both are members of the TNF receptor family which also includeTNFR-2, low affinity NGFR, CD40, and CD30, among others (C. A. Smith, etal., Science 248, 1019-23 (1990); M. Tewari, et al., in Modular Texts inMolecular and Cell Biology M. Purton, Heldin, Carl, Ed. (Chapman andHall, London, 1995). While family members are defined by the presence ofcysteine-rich repeats in their extracellular domains, Fas/APO-1 andTNFR-1 also share a region of intracellular homology, appropriatelydesignated the “death domain”, which is distantly related to theDrosophila suicide gene, reaper (P. Golstein, et al., Cell 81, 185-6(1995); K. White et al., Science 264, 677-83 (1994)). This shared deathdomain suggests that both receptors interact with a related set ofsignal transducing molecules that, until recently, remainedunidentified. Activation of Fas/APO-1 recruits the deathdomain-containing adapter molecule FADD/MORT1 (A. M. Chinnaiyan, et al.,Cell 81, 505-12 (1995); M. P. Boldin, et al., J. Biol Chem 270, 7795-8(1995); F. C. Kischkel, et al., EMBO 14, 5579-5588 (1995)), which inturn binds and presumably activates FLICE/MACH1, a member of theICE/CED-3 family of pro-apoptotic proteases (M. Muzio et al., Cell 85,817-827 (1996); M. P. Boldin, et al., Cell 85, 803-815 (1996)). Whilethe central role of Fas/APO-1 is to trigger cell death, TNFR-1 cansignal an array of diverse biological activities-many of which stem fromits ability to activate NF-kB (L. A. Tartaglia, et al., Immunol Today13, 151-3 (1992)). Accordingly, TNFR-1 recruits the multivalent adaptermolecule TRADD, which like FADD, also contains a death domain (H. Hsu,et al., Cell 81, 495-504 (1995); H. Hsu, et al., Cell 84, 299-308(1996)). Through its associations with a number of signaling moleculesincluding FADD, TRAF2, and RIP, TRADD can signal both apoptosis andNF-kB activation (H. Hsu, et al., Cell 84, 299-308 (1996); H. Hsu, etal., Immunity 4, 387-396 (1996)). Recently a new apoptosis inducingligand was discovered. Wiley, S. R. et al., refer to the new molecule asTNF-related apoptosis-inducing ligand or (TRAIL) (Immunity 3:673-682(1995)). Pitti, R. M. et al., refer to the new molecule as Apo-2 ligandor (iApo-2Lî). This molecule was also disclosed in copending U.S.Provisional Patent Application Serial No. 60/013405. For convenience, itwill be referred to herein as TRAIL. Unlike FAS ligand whose transcriptsappear to be largely restricted to stimulated T-cells, significantlevels of TRAIL are seen in many tissues, and it is constitutivelytranscribed by some cell lines. It has been shown that TRAIL actsindependently from FAS ligand (Wiley, S. R., et al. (1995)), supra).Studies by Marsters, S. A. et al., have indicated that TRAIL activatesapoptosis rapidly, within a time frame that is similar to deathsignalling by FAS/Apo-1L but much faster than TNF-induced apoptosis(Current Biology, 6:750-752 (1996)). All work to date suggest that thereceptor for TRAIL is not one of the many known TNF-receptors.

[0134] Based upon the localization of the protein of the presentinvention in mitochondria and its apoptosis-dependent translocation tothe nucleus, it is clear that this protein is useful in treating and/orpreventing a number of diseases and/or disorders, particularlyproliferative conditions and would again be useful in modulating theimmune response to transformed cells and aberrant phenotypes.Furthermore, the protein may also be used to determine biologicalactivity, to raise antibodies, as tissue markers, to isolate cognateligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

[0135] The potential for the polynucleotides and/or polypeptides of thepresent invention to elicit an apoptotic response can be assayed througha number of means, particular through the application of cell deathassays. Briefly, cell death assays are performed essentially aspreviously described (A. M. Chinnaiyan, et al., Cell 81, 505-12 (1995);M. P. Boldin, et al., J Biol Chem 270, 7795-8 (1995); F. C. Kischkel, etal., EMBO 14, 5579-5588 (1995); A. M. Chinnaiyan, et al., J Biol Chem271, 4961-4965 (1996)).

[0136] Briefly, MCF-7 human breast carcinoma clonal cell lines stablytransfected with either vector alone or a CrmA expression construct (M.Tewari, et al., J Biol Chem 270, 3255-60 (1995)), are transientlytransfected with pCMV-AIF-galatosidase (or pCMV-AIF-galactosidase aninactive form of AIF) in the presence of a ten-fold excess of pcDNA3expression constructs encoding the indicated proteins usinglipofectamine (GIBCO-BRL). 293 cells are likewise transfected using theCaPO4 method. The ICE family inhibitor z-VAD-fmk (Enzyme SystemsProducts, Dublin, Calif.) is added to the cells at a concentration of 10μM, 5 hrs after transfection. 32 hours following transfection, cells arefixed and stained with X-Gal as previously described (A. M. Chinnaiyan,et al., Cell 81, 505-12 (1995); M. P. Boldin, et al., J Biol Chem 270,7795-8 (1995); F. C. Kischkel, et al., EMBO 14, 5579-5588 (1995)). TABLE1 NT AA ATCC SEQ 5′NT 3′NT 5′NT SEQ Last Deposit ID Total of of of ID AAGene CDNA No:Z NO NT Clone Clone Start NO: of No. Clone ID and DateVector X Seq. Seq. Seq. Codon Y ORF 1 HLDOK36 PTA161 pCMVSport 2 2045 12045 187 11 304 Dec. 1, 1999 3.0 2 HDPBW68 PTAI61 pCMVSport 3 4415 14415 425 12 953 Dec. 1, 1999 3.0 2 HDPBW68 PTAI61 pCMVSport 10 3789 13789 558 19 705 Dec. 1, 1999 3.0 3 HHEFO24 PTA161 pCMVSport 4 2066 12066 12 13 445 Dec. 1, 1999 3.0 4 HEGAL46 PTA161 Uni-ZAP XR 5 1406 11406 237 14 340 Dec. 1, 1999 5 HFOYCO2 PTA161 pSport1 6 3172 372 2212252 15 317 Dec. 1, 1999 6 HDABV82 PTA161 pSport1 7 2290 1 2290 222 16 90Dec. 1, 1999 7 HSVAF16 PTA161 Uni-ZAP XR 8 1316 101 1316 524 17 216 Dec.1, 1999 8 HSJFO61 PTA181 Uni-ZAP XR 9 2150 647 2076 95 18 613 Dec. 7,1999

[0137] Table 1 summarizes the information corresponding to each “GeneNo:” described above. The nucleotide sequence identified as “NT SEQ IDNO:X” was assembled from partially homologous (“overlapping”) sequencesobtained from the “cDNA clone ID” identified in Table 1 and, in somecases, from additional related DNA clones. The overlapping sequenceswere assembled into a single contiguous sequence of high redundancy(usually three to five overlapping sequences at each nucleotideposition), resulting in a final sequence identified as SEQ ID NO:X.

[0138] The cDNA Clone ID was deposited on the date and given thecorresponding deposit number listed in “ATCC Deposit No:Z and Date.”Some of the deposits contain multiple different clones corresponding tothe same gene. “Vector” refers to the type of vector contained in thecDNA Clone ID.

[0139] “Total NT Seq.” refers to the total number of nucleotides in thecontig identified by “Gene No:”. The deposited plasmid contains all ofthese sequences, reflected by the tide position indicated as “5′ NT ofClone Seq.” and the “3′ NT of Clone Seq.” of SEQ ID NO:X. The nucleotideposition of SEQ ID NO:X of the putative methionine start codon (ifpresent) is identified as “5′ NT of Start Codon.” Similarly, thenucleotide position of SEQ ID NO:X of the predicted signal sequence (ifpresent) is identified as “5′ NT of First AA of Signal Pep.”

[0140] The translated amino acid sequence, beginning with the firsttranslated codon of the polynucleotide sequence, is identified as “AASEQ ID NO:Y,” although other reading frames can also be easilytranslated using known molecular biology techniques. The polypeptidesproduced by these alternative open reading frames are specificallycontemplated by the present invention.

[0141] SEQ ID NO:X (where X may be any of the polynucleotide sequencesdisclosed in the sequence listing) and the translated SEQ ID NO:Y (whereY may be any of the polypeptide sequences disclosed in the sequencelisting) are sufficiently accurate and otherwise suitable for a varietyof uses well known in the art and described further below. For instance,SEQ ID NO:X has uses including, but not limited to, in designing nucleicacid hybridization probes that will detect nucleic acid sequencescontained in SEQ ID NO:X or the cDNA contained in a deposited plasmid.These probes will also hybridize to nucleic acid molecules in biologicalsamples, thereby enabling a variety of forensic and diagnostic methodsof the invention. Similarly, polypeptides identified from SEQ ID NO:Yhave uses that include, but are not limited to generating antibodies,which bind specifically to the secreted proteins encoded by the cDNAclones identified in Table 1.

[0142] Nevertheless, DNA sequences generated by sequencing reactions cancontain sequencing errors. The errors exist as misidentifiednucleotides, or as insertions or deletions of nucleotides in thegenerated DNA sequence. The erroneously inserted or deleted nucleotidescause frame shifts in the reading frames of the predicted amino acidsequence. In these cases, the predicted amino acid sequence divergesfrom the actual amino acid sequence, even though the generated DNAsequence may be greater than 99.9% identical to the actual DNA sequence(for example, one base insertion or deletion in an open reading frame ofover 1000 bases).

[0143] Accordingly, for those applications requiring precision in thenucleotide sequence or the amino acid sequence, the present inventionprovides not only the generated nucleotide sequence identified as SEQ IDNO:X, and the predicted translated amino acid sequence identified as SEQID NO:Y, but also a sample of plasmid DNA containing a human cDNA of theinvention deposited with the ATCC, as set forth in Table 1. Thenucleotide sequence of each deposited plasmid can readily be determinedby sequencing the deposited plasmid in accordance with known methods.

[0144] The predicted amino acid sequence can then be verified from suchdeposits. Moreover, the amino acid sequence of the protein encoded by aparticular plasmid can also be directly determined by peptide sequencingor by expressing the protein in a suitable host cell containing thedeposited human cDNA, collecting the protein, and determining itssequence.

[0145] Also provided in Table 1 is the name of the vector which containsthe cDNA plasmid. Each vector is routinely used in the art. Thefollowing additional information is provided for convenience.

[0146] Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636),Uni-Zap XR (U.S. Pat. Nos. 5,128,256 and 5,286,636), Zap Express (U.S.Pat. Nos. 5,128,256 and 5,286,636), pBluescript (pBS) (Short, J. M. etal., Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. andShort, J. M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees,M. A. et al., Strategies 5:58-61 (1992)) are commercially available fromStratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla,Calif., 92037. pBS contains an ampicillin resistance gene and pBKcontains a neomycin resistance gene. Phagemid pBS may be excised fromthe Lambda Zap and Uni-Zap XR vectors, and phagemid pBK may be excisedfrom the Zap Express vector. Both phagemids may be transformed into E.coli strain XL-1 Blue, also available from Stratagene.

[0147] Vectors pSport1, pCMVSport 1.0, pCMVSport 2.0 and pCMVSport 3.0,were obtained from Life Technologies, Inc., P. O. Box 6009,Gaithersburg, Md. 20897. All Sport vectors contain an ampicillinresistance gene and may be transformed into E. coli strain DH10B, alsoavailable from Life Technologies. See, for instance, Gruber, C. E., etal., Focus 15:59 (1993). Vector lafmid BA (Bento Soares, ColumbiaUniversity, New York, N.Y.) contains an ampicillin resistance gene andcan be transformed into E. coli strain XL-1 Blue. Vector pCR®2.1, whichis available from Invitrogen, 1600 Faraday Avenue, Carlsbad, Calif.92008, contains an ampicillin resistance gene and may be transformedinto E. coli strain DH10B, available from Life Technologies. See, forinstance, Clark, J. M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D.et al., Bio/Technology 9: (1991).

[0148] The present invention also relates to the genes corresponding toSEQ ID NO:X, SEQ ID NO:Y, and/or a deposited plasmid (cDNA plasmid:Z).The corresponding gene can be isolated in accordance with known methodsusing the sequence information disclosed herein. Such methods include,but are not limited to, preparing probes or primers from the disclosedsequence and identifying or amplifying the corresponding gene fromappropriate sources of genomic material.

[0149] Also provided in the present invention are allelic variants,orthologs, and/or species homologs. Procedures known in the art can beused to obtain full-length genes, allelic variants, splice variants,full-length poding portions, orthologs, and/or species homologs of genescorresponding to SEQ ID NO:X, SEQ ID NO:Y, and/or cDNA plasmid:Z, usinginformation from the sequences disclosed herein or the clones depositedwith the ATCC. For example, allelic variants and/or species homologs maybe isolated and identified by making suitable probes or primers from thesequences provided herein and screening a suitable nucleic acid sourcefor allelic variants and/or the desired homologue.

[0150] The present invention provides a polynucleotide comprising, oralternatively consisting of, the nucleic acid sequence of SEQ ID NO:Xand/or cDNA plasmid:Z. The present invention also provides a polypeptidecomprising, or alternatively, consisting of, the polypeptide sequence ofSEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X, and/or a polypeptideencoded by the cDNA in cDNA plasmid:Z. Polynucleotides encoding apolypeptide comprising, or alternatively consisting of the polypeptidesequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X and/or apolypeptide encoded by the cDNA in cDNA plasmid:Z, are also encompassedby the invention. The present invention further encompasses apolynucleotide comprising, or alternatively consisting of the complementof the nucleic acid sequence of SEQ ID NO:X, and/or the complement ofthe coding strand of the cDNA in cDNA plasmid:Z.

[0151] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases and mayhave been publicly available prior to conception of the presentinvention. Preferably, such related polynucleotides are specificallyexcluded from the scope of the present invention. To list every relatedsequence would unduly burden the disclosure of this application.Accordingly, preferably excluded from SEQ ID NO:X are one or morepolynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 and the finalnucleotide minus 15 of SEQ IID NO:X, b is an integer of 15 to the finalnucleotide of SEQ ID NO:X, where both a and b correspond to thepositions of nucleotide residues shown in SEQ ID NO:X, and where b isgreater than or equal to a+14.

[0152] RACE Protocol For Recovery of Full-Length Genes

[0153] Partial cDNA clones can be made full-length by utilizing therapid amplification of cDNA ends (RACE) procedure described in Frohman,M. A., et al., Proc. Nat'l. Acad. Sci. USA, 85:8998-9002 (1988). A cDNAclone missing either the 5′ or 3′ end can be reconstructed to includethe absent base pairs extending to the translational start or stopcodon, respectively. In some cases, cDNAs are missing the start oftranslation, therefor. The following briefly describes a modification ofthis original 5′ RACE procedure. Poly A+ or total RNA is reversetranscribed with Superscript II (Gibco/BRL) and an antisense orcomplementary primer specific to the cDNA sequence. The primer isremoved from the reaction with a Microcon Concentrator (Amicon). Thefirst-strand cDNA is then tailed with DATP and terminal deoxynucleotidetransferase (Gibco/BRL). Thus, an anchor sequence is produced which isneeded for PCR amplification. The second strand is synthesized from theda-tail in PCR buffer, Taq DNA polymerase (Perkin-Elmer Cetus), anoligo-dT primer containing three adjacent restriction sites (XhoI, SalIand ClaI) at the 5′ end and a primer containing just these restrictionsites. This double-stranded cDNA is PCR amplified for 40 cycles with thesame primers as well as a nested cDNA-specific antisense primer. The PCRproducts are size-separated on an ethidium bromide-agarose gel and theregion of gel containing cDNA products the predicted size of missingprotein-coding DNA is removed. cDNA is purified from the agarose withthe Magic PCR Prep kit (Promega), restriction digested with XhoI orSalI, and ligated to a plasmid such as pBluescript SKII (Stratagene) atXhoI and EcoRV sites. This DNA is transformed into bacteria and theplasmid clones sequenced to identify the correct protein-coding inserts.Correct 5′ ends are confirmed by comparing this sequence with theputatively identified homologue and overlap with the partial cDNA clone.Similar methods known in the art and/or commercial kits are used toamplify and recover 3′ ends.

[0154] Several quality-controlled kits are commercially available forpurchase. Similar reagents and methods to those above are supplied inkit form from Gibco/BRL for both 5′ and 3′ RACE for recovery of fulllength genes. A second kit is available from Clontech which is amodification of a related technique, SLIC (single-stranded ligation tosingle-stranded cDNA), developed by Dumas et al., Nucleic Acids Res.,19:5227-32 (1991). The major differences in procedure are that the RNAis alkaline hydrolyzed after reverse transcription and RNA ligase isused to join a restriction site-containing anchor primer to thefirst-strand cDNA. This obviates the necessity for the dA-tailingreaction which results in a polyT stretch that is difficult to sequencepast.

[0155] An alternative to generating 5′ or 3′ cDNA from RNA is to usecDNA library double-stranded DNA. An asymmetric PCR-amplified antisensecDNA strand is synthesized with an antisense cDNA-specific primer and aplasmid-anchored primer. These primers are removed and a symmetric PCRreaction is performed with a nested cDNA-specific antisense primer andthe plasmid-anchored primer.

[0156] RNA Ligase Protocol for Generating the 5′ or 3′ End Sequences toObtain Full Length Genes

[0157] Once a gene of interest is identified, several methods areavailable for the identification of the 5′ or 3′ portions of the genewhich may not be present in the original cDNA plasmid. These methodsinclude, but are not limited to, filter probing, clone enrichment usingspecific probes and protocols similar and identical to 5′ and 3′RACE.While the full length gene may be present in the library and can beidentified by probing, a useful method for generating the 5′ or 3′ endis to use the existing sequence information from the original cDNA togenerate the missing information. A method similar to 5′RACE isavailable for generating the missing 5′ end of a desired full-lengthgene. (This method was published by Fromont-Racine et al., Nucleic AcidsRes., 21(7):1683-1684 (1993)). Briefly, a specific RNA oligonucleotideis ligated to the 5′ ends of a population of RNA presumably containingfull-length gene RNA transcript and a primer set containing a primerspecific to the ligated RNA oligonucleotide and a primer specific to aknown sequence of the gene of interest, is used to PCR amplify the 5′portion of the desired full length gene which may then be sequenced andused to generate the full length gene. This method starts with total RNAisolated from the desired source, poly A RNA may be used but is not aprerequisite for this procedure. The RNA preparation may then be treatedwith phosphatase if necessary to eliminate 5′ phosphate groups ondegraded or damaged RNA which may interfere with the later RNA ligasestep. The phosphatase if used is then inactivated and the RNA is treatedwith tobacco acid pyrophosphatase in order to remove the cap structurepresent at the 5′ ends of messenger RNAs. This reaction leaves a 5′phosphate group at the 5′ end of the cap cleaved RNA which can then beligated to an RNA oligonucleotide using T4 RNA ligase. This modified RNApreparation can then be used as a template for first strand cDNAsynthesis using a gene specific oligonucleotide. The first strandsynthesis reaction can then be used as a template for PCR amplificationof the desired 5′ end using a primer specific to the ligated RNAoligonucleotide and a primer specific to the known sequence of theapoptosis related gene of interest. The resultant product is thensequenced and analyzed to confirm that the 5′ end sequence belongs tothe relevant apoptosis related gene.

[0158] Polynucleotide and Polypeptide Fragments

[0159] The present invention is also directed to polynucleotidefragments of the polynucleotides (nucleic acids) of the invention. Inthe present invention, a “polynucleotide fragment” refers to apolynucleotide having a nucleic acid sequence which: is a portion of thecDNA contained in cDNA plasmid:Z or encoding the polypeptide encoded bythe cDNA contained in cDNA plasmid:Z; is a portion of the polynucleotidesequence in SEQ ID NO:X or the complementary strand thereto; is apolynucleotide sequence encoding a portion of the polypeptide of SEQ IDNO:Y; or is a polynucleotide sequence encoding a portion of apolypeptide encoded by SEQ ID NO:X. The nucleotide fragments of theinvention are preferably at least about 15 nt, and more preferably atleast about 20 nt, still more preferably at least about 30 nt, and evenmore preferably, at least about 40 nt, at least about 50 nt, at leastabout 75 nt, at least about 100 nt, at least about 125 nt, or at leastabout 150 nt in length. A fragment “at least 20 nt in length,” forexample, is intended to include 20 or more contiguous bases from, forexample, the sequence contained in the cDNA in cDNA plasmid:Z, or thenucleotide sequence shown in SEQ ID NO:X or the complementary standthereto. In this context “about” includes the particularly recitedvalue, or a value larger or smaller by several (5, 4, 3, 2, or 1)nucleotides. These nucleotide fragments have uses that include, but arenot limited to, as diagnostic probes and primers as discussed herein. Ofcourse, larger fragments (e.g., at least 150, 175, 200, 250, 500, 600,1000, or 2000 nucleotides in length ) are also encompassed by theinvention.

[0160] Moreover, representative examples of polynucleotide fragments ofthe invention, include, for example, fragments comprising, oralternatively consisting of, a sequence from about nucleotide number1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400,401-450, 451-500, 501-550, 551-600, 651-700,701-750, 751-800, 800-850,851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200,1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500,1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800,1801-1850, 1851-1900, 1901-1950, 1951-2000, 2001-2050, 2051-2100,2101-2150, 2151-2200, 2201-2250, 2251-2300, 2301-2350, 2351-2400,2401-2450, 2451-2500, 2501-2550, 2551-2600, 2601-2650, 2651-2700,2701-2750, 2751-2800, 2801-2850, 2851-2900, 2901-2950,.2951-3000,3001-3050, 3051-3100, 3101-3150, 3151-3200, 3201-3250, 3251-3300,3301-3350, 3351-3400, 3401-3450, 3451-3500, 3501-3550, 3551-3600,3601-3650, 3651-3700, 3701-3750, 3751-3800, 3801-3850, 3851-3900,3901-3950, 3951-4000, 4001-4050, 4051-4100, 4101-4150, 4151-4200,4201-4250, 4251-4300, 4301-4350, 4351-4400, and/or 4401-4415 of SEQ IDNO:X, or the complementary strand thereto. In this context “about”includes the particularly recited range or a range larger or smaller byseveral (5, 4, 3, 2, or 1) nucleotides, at either terminus or at bothtermini. Preferably, these fragments encode a polypeptide which has afunctional activity (e.g. biological activity) of the polypeptideencoded by a polynucleotide of which the sequence is a portion. Morepreferably, these fragments can be used as probes or primers asdiscussed herein. Polynucleotides which hybridize to one or more ofthese fragments under stringent hybridization conditions oralternatively, under lower stringency conditions, are also encompassedby the invention, as are polypeptides encoded by these polynucleotidesor fragments.

[0161] Moreover, representative examples of polynucleotide fragments ofthe invention, include, for example, fragments comprising, oralternatively consisting of, a sequence from about nucleotide number1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400,401-450, 451-500, 501-550, 551-600, 651-700,701-750, 751-800, 800-850,851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200,1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500,1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800,1801-1850, 1851-1900, 1901-1950, 1951-2000, 2001-2050, 2051-2100,2101-2150, 2151-2200, 2201-2250, 2251-2300, 2301-2350, 2351-2400,2401-2450, 2451-2500, 2501-2550, 2551-2600, 2601-2650, 2651-2700,2701-2750, 2751-2800, 2801-2850, 2851-2900, 2901-2950, 2951-3000,3001-3050, 3051-3100, 3101-3150, 3151-3200, 3201-3250, 3251-3300,3301-3350, 3351-3400, 3401-3450, 3451-3500, 3501-3550, 3551-3600,3601-3650, 3651-3700, 3701-3750, 3751-3800, 3801-3850, 3851-3900,3901-3950, 3951-4000, 4001-4050, 4051-4100, 4101-4150, 4151-4200,4201-4250, 4251-4300, 4301-4350, 4351-4400, and/or 4401-4415 of the cDNAnucleotide sequence contained in cDNA plasmid:Z, or the complementarystrand thereto. In this context “about” includes the particularlyrecited range or a range larger or smaller by several (5, 4, 3, 2, or 1)nucleotides, at either terminus or at both termini. Preferably, thesefragments encode a polypeptide which has a functional activity (e.g.biological activity) of the polypeptide encoded by the cDNA nucleotidesequence contained in cDNA plasmid:Z. More preferably, these fragmentscan be used as probes or primers as discussed herein. Polynucleotideswhich hybridize to one or more of these fragments under stringenthybridization conditions, or alternatively, under lower stringencyconditions are also encompassed by the invention, as are polypeptidesencoded by these polynucleotides or fragments.

[0162] In the present invention, a “polypeptide fragment” refers to anamino acid sequence which is a portion of that contained in SEQ ID NO:Y,a portion of an amino acid sequence encoded by the polynucleotidesequence of SEQ ID NO:X, and/or encoded by the cDNA in cDNA plasmid:Z.Protein (polypeptide) fragments may be “free-standing,” or comprisedwithin a larger polypeptide of which the fragment forms a part orregion, most preferably as a single continuous region. Representativeexamples of polypeptide fragments of the invention, include, forexample, fragments comprising, or alternatively consisting of, an aminoacid sequence from about amino acid number 1-20, 21-40, 41-60, 61-80,81-100, 102-120, 121-140, 141-160, 161-180, 181-200, 201-220, 221-240,241-260, 261-280, 281-300, 301-320, 321-340, 341-360, 361-380, 381-400,401-420, 421-440, 441-460, 461-480, 481-500, 501-520, 521-540, 541-560,561-580, 581-600, 601-620, 621-640, 641-660, 661-680, 681-700, 701-720,721-740, 741-760, 761-780, 781-800, 801-820, 821-840, 841-860, 861-880,881-900, 901-920, 921-940, and/or 941-953 of the coding region of SEQ IDNO:Y. Moreover, polypeptide fragments of the invention may be at leastabout 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 100, 110, 120, 130, 140, or 150 amino acids in length. In thiscontext “about” includes the particularly recited ranges or values, orranges or values larger or smaller by several (5, 4, 3, 2, or 1) aminoacids, at either terminus or at both termini. Polynucleotides encodingthese polypeptide fragments are also encompassed by the invention.

[0163] Even if deletion of one or more amino acids from the N-terminusof a protein results in modification of loss of one or more biologicalfunctions of the protein, other functional activities (e.g., biologicalactivities, ability to multimerize, ability to bind a ligand) may stillbe retained. For example, the ability of shortened muteins to induceand/or bind to antibodies which recognize the complete or mature formsof the polypeptides generally will be retained when less than themajority of the residues of the complete or mature polypeptide areremoved from the N-terminus. Whether a particular polypeptide lackingN-terminal residues of a complete polypeptide retains such immunologicactivities can readily be determined by routine methods described hereinand otherwise known in the art. It is not unlikely that a mutein with alarge number of deleted N-terminal amino acid residues may retain somebiological or immunogenic activities. In fact, peptides composed of asfew as six amino acid residues may often evoke an immune response.

[0164] Accordingly, polypeptide fragments of the invention include thesecreted protein as well as the mature form. Further preferredpolypeptide fragments include the secreted protein or the mature formhaving a continuous series of deleted residues from the amino or thecarboxy terminus, or both. For example, any number of amino acids,ranging from 1-60, can be deleted from the amino terminus of either thesecreted polypeptide or the mature form. Similarly, any number of aminoacids, ranging from 1-30, can be deleted from the carboxy terminus ofthe secreted protein or mature form. Furthermore, any combination of theabove amino and carboxy terminus deletions are preferred. Similarly,polynucleotides encoding these polypeptide fragments are also preferred.

[0165] The present invention further provides polypeptides having one ormore residues deleted from the amino terminus of the amino acid sequenceof a polypeptide disclosed herein (e.g., a polypeptide of SEQ ID NO:Y, apolypeptide encoded by the polynucleotide sequence contained in SEQ IDNO:X, and/or a polypeptide encoded by the cDNA contained in cDNAplasmid:Z). In particular, N-terminal deletions may be described by thegeneral formula m-q, where q is a whole integer representing the totalnumber of amino acid residues in a polypeptide of the invention (e.g.,the polypeptide disclosed in SEQ ID NO:Y), and m is defined as anyinteger ranging from 2 to q-6. Polynucleotides encoding thesepolypeptides, including fragments and/or variants, are also encompassedby the invention.

[0166] Also as mentioned above, even if deletion of one or more aminoacids from the C-terminus of a protein results in modification of lossof one or more biological functions of the protein, other functionalactivities (e.g., biological activities, ability to multimerize, abilityto bind a ligand) may still be retained. For example the ability of theshortened mutein to induce and/or bind to antibodies which recognize thecomplete or mature forms of the polypeptide generally will be retainedwhen less than the majority of the residues of the complete or maturepolypeptide are removed from the C-terminus. Whether a particularpolypeptide lacking C-terminal residues of a complete polypeptideretains such immunologic activities can readily be determined by routinemethods described herein and otherwise known in the art. It is notunlikely that a mutein with a large number of deleted C-terminal aminoacid residues may retain some biological or immunogenic activities. Infact, peptides composed of as few as six amino acid residues may oftenevoke an immune response.

[0167] Accordingly, the present invention further provides polypeptideshaving one or more residues from the carboxy terminus of the amino acidsequence of a polypeptide disclosed herein (e.g., a polypeptide of SEQID NO:Y, a polypeptide encoded by the polynucleotide sequence containedin SEQ ID NO:X, and/or a polypeptide encoded by the cDNA contained incDNA plasmid:Z). In particular, C-terminal deletions may be described bythe general formula 1-n, where n is any whole integer ranging from 6 toq-1, and where n corresponds to the position of an amino acid residue ina polypeptide of the invention. Polynucleotides encoding thesepolypeptides, including fragments and/or variants, are also encompassedby the invention.

[0168] In addition, any of the above described N- or C-terminaldeletions can be combined to produce a N- and C-terminal deletedpolypeptide. The invention also provides polypeptides having one or moreamino acids deleted from both the amino and the carboxyl termini, whichmay be described generally as having residues m-n of a polypeptideencoded by SEQ ID NO:X (e.g., including, but not limited to, thepreferred polypeptide disclosed as SEQ ID NO:Y), and/or the cDNA in cDNAplasmid:Z, and/or the complement thereof, where n and m are integers asdescribed above. Polynucleotides encoding these polypeptides, includingfragments and/or variants, are also encompassed by the invention.

[0169] Any polypeptide sequence contained in the polypeptide of SEQ IDNO:Y, encoded by the polynucleotide sequences set forth as SEQ ID NO:X,or encoded by the cDNA in cDNA plasmid:Z may be analyzed to determinecertain preferred regions of the polypeptide. For example, the aminoacid sequence of a polypeptide encoded by a polynucleotide sequence ofSEQ ID NO:X or the cDNA in cDNA plasmid:Z may be analyzed using thedefault parameters of the DNASTAR computer algorithm (DNASTAR, Inc.,1228 S. Park St., Madison, Wis. 53715 USA; http://www.dnastar.com/).

[0170] Polypeptide regions that may be routinely obtained using theDNASTAR computer algorithm include, but are not limited to,Gamier-Robson alpha-regions, beta-regions, turn-regions, andcoil-regions, Chou-Fasman alpha-regions, beta-regions, and tum-regions,Kyte-Doolittle hydrophilic regions and hydrophobic regions, Eisenbergalpha- and beta-amphipathic regions, Karplus-Schulz flexible regions,Emini surface-forming regions and Jameson-Wolf regions of high antigenicindex. Among highly preferred polynucleotides of the invention in thisregard are those that encode polypeptides comprising regions thatcombine several structural features, such as several (e.g., 1, 2, 3 or4) of the features set out above.

[0171] Additionally, Kyte-Doolittle hydrophilic regions and hydrophobicregions, Emini surface-forming regions, and Jameson-Wolf regions of highantigenic index (i.e., containing four or more contiguous amino acidshaving an antigenic index of greater than or equal to 1.5, as identifiedusing the default parameters of the Jameson-Wolf program) can routinelybe used to determine polypeptide regions that exhibit a high degree ofpotential for antigenicity. Regions of high antigenicity are determinedfrom data by DNASTAR analysis by choosing values which represent regionsof the polypeptide which are likely to be exposed on the surface of thepolypeptide in an environment in which antigen recognition may occur inthe process of initiation of an immune response.

[0172] Preferred polypeptide fragments of the invention are fragmentscomprising, or alternatively, consisting of, an amino acid sequence thatdisplays a functional activity (e.g. biological activity) of thepolypeptide sequence of which the amino acid sequence is a fragment. Bya polypeptide displaying a “functional activity” is meant a polypeptidecapable of one or more known functional activities associated with afull-length protein, such as, for example, biological activity,antigenicity, immunogenicity, and/or multimerization, as describedsupra.

[0173] Other preferred polypeptide fragments are biologically activefragments. Biologically active fragments are those exhibiting activitysimilar, but not necessarily identical, to an activity of thepolypeptide of the present invention. The biological activity of thefragments may include an improved desired activity, or a decreasedundesirable activity.

[0174] In preferred embodiments, polypeptides of the invention comprise,or alternatively consist of, one, two, three, four, five or more of theantigenic fragments of the polypeptide of SEQ ID NO:Y, or portionsthereof. Polynucleotides encoding these polypeptides, includingfragments and/or variants, are also encompassed by the invention.

[0175] The present invention encompasses polypeptides comprising, oralternatively consisting of, an epitope of the polypeptide sequenceshown in SEQ ID NO:Y, or an epitope of the polypeptide sequence encodedby the cDNA in cDNA plasmid:Z, or encoded by a polynucleotide thathybridizes to the complement of an epitope encoding sequence of SEQ IDNO:X, or an epitope encoding sequence contained in cDNA plasmid:Z understringent hybridization conditions, or alternatively, under lowerstringency hybridization, as defined supra. The present inventionfurther encompasses polynucleotide sequences encoding an epitope of apolypeptide sequence of the invention (such as, for. example, thesequence disclosed in SEQ ID NO:X), polynucleotide sequences of thecomplementary strand of a polynucleotide sequence encoding an epitope ofthe invention, and polynucleotide sequences which hybridize to thiscomplementary strand under stringent hybridization conditions, oralternatively, under lower stringency hybridization conditions, asdefined supra.

[0176] The term “epitopes,” as used herein, refers to portions of apolypeptide having antigenic or immunogenic activity in an animal,preferably a mammal, and most preferably in a human. In a preferredembodiment, the present invention encompasses a polypeptide comprisingan epitope, as well as the polynucleotide encoding this polypeptide. An“immunogenic epitope,” as used herein, is defined as a portion of aprotein that elicits an antibody response in an animal, as determined byany method known in the art, for example, by the methods for generatingantibodies described infra. (See, for example, Geysen et al., Proc.Natl. Acad. Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,”as used herein, is defined as a portion of a protein to which anantibody can immunospecifically bind its antigen as determined by anymethod well known in the art, for example, by the immunoassays describedherein. Immunospecific binding excludes non-specific binding but doesnot necessarily exclude cross-reactivity with other antigens. Antigenicepitopes need not necessarily be immunogenic.

[0177] Fragments which function as epitopes may be produced by anyconventional means. (See, e.g., Houghten, R. A., Proc. Natl. Acad. Sci.USA 82:5131-5135 (1985) further described in U.S. Pat. No. 4,631,211.)

[0178] In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 20, at least 25, atleast 30, at least 40, at least 50, and, most preferably, between about15 to about 30 amino acids. Preferred polypeptides comprisingimmunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acidresidues in length. Additional non-exclusive preferred antigenicepitopes include the antigenic epitopes disclosed herein, as well asportions thereof. Antigenic epitopes are useful, for example, to raiseantibodies, including monoclonal antibodies, that specifically bind theepitope. Preferred antigenic epitopes include the antigenic epitopesdisclosed herein, as well as any combination of two, three, four, fiveor more of these antigenic epitopes. Antigenic epitopes can be used asthe target molecules in immunoassays. (See, for instance, Wilson et al.,Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).

[0179] Similarly, immunogenic epitopes can be used, for example, toinduce antibodies according to methods well known in the art. (See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al.,Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol.66:2347-2354 (1985). Preferred immunogenic epitopes include theimmunogenic epitopes disclosed herein, as well as any combination oftwo, three, four, five or more of these immunogenic epitopes. Thepolypeptides comprising one or more immunogenic epitopes may bepresented for eliciting an antibody response together with a carrierprotein, such as an albumin, to an animal system (such as rabbit ormouse), or, if the polypeptide is of sufficient length (at least about25 amino acids), the polypeptide may be presented without a carrier.However, immunogenic epitopes comprising as few as 8 to 10 amino acidshave been shown to be sufficient to raise antibodies capable of bindingto, at the very least, linear epitopes in a denatured polypeptide (e.g.,in Western blotting).

[0180] Epitope-bearing polypeptides of the present invention may be usedto induce antibodies according to methods well known in the artincluding, but not limited to, in vivo immunization, in vitroimmunization, and phage display methods. See, e.g., Sutcliffe et al.,supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol.,66:2347-2354 (1985). If in vivo immunization is used, animals may beimmunized with free peptide; however, anti-peptide antibody titer may beboosted by coupling the peptide to a macromolecular carrier, such askeyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance,peptides containing cysteine residues may be coupled to a carrier usinga linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),while other peptides may be coupled to carriers using a more generallinking agent such as glutaraldehyde. Animals such as rabbits, rats andmice are immunized with either free or carrier-coupled peptides, forinstance, by intraperitoneal and/or intradermal injection of emulsionscontaining about 100 μg of peptide or carrier protein and Freund'sadjuvant or any other adjuvant known for stimulating an immune response.Several booster injections may be needed, for instance, at intervals ofabout two weeks, to provide a useful titer of anti-peptide antibodywhich can be detected, for example, by ELISA assay using freepeptide.adsorbed to a solid surface. The titer of anti-peptideantibodies in serum from an immunized animal may be increased byselection of anti-peptide antibodies, for instance, by adsorption to thepeptide on a solid support and elution of the selected antibodiesaccording to methods well known in the art.

[0181] As one of skill in the art will appreciate, and as discussedabove, the polypeptides of the present invention and immunogenic and/orantigenic epitope fragments thereof can be fused to other polypeptidesequences. For example, the polypeptides of the present invention may befused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM),or portions thereof (CH1, CH2, CH3, or any combination thereof andportions thereof) resulting in chimeric polypeptides. Such fusionproteins may facilitate purification and may increase half-life in vivo.This has been shown for chimeric proteins consisting of the first twodomains of the human CD4-polypeptide and various domains of the constantregions of the heavy or light chains of mammalian immunoglobulins. See,e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988). Enhanceddelivery of an antigen across the epithelial barrier to the immunesystem has been demonstrated for antigens (e.g., insulin) conjugated toan FcRn binding partner such as IgG or Fc fragments (see, e.g., PCTPublications WO 96/22024 and WO 99/04813). IgG Fusion proteins that havea disulfide-linked dimeric structure due to the IgG portion desulfidebonds have also been found to be more efficient in binding andneutralizing other molecules than monomeric polypeptides or fragmentsthereof alone. See, e.g., Fountoulakis et al., J. Biochem.,270:3958-3964 (1995).

[0182] Similarly, EP-A-O 464 533 (Canadian counterpart 2045869)discloses fusion proteins comprising various portions of constant regionof immunoglobulin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP-A 0232 262.) Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, may be desired. For example, the Fc portion may hinder therapyand diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johansonet al., J. Biol. Chem. 270:9459-9471 (1995).)

[0183] Moreover, the polypeptides of the present invention can be fusedto marker sequences, such as a peptide which facilitates purification ofthe fused polypeptide. In preferred embodiments, the marker amino acidsequence is a hexa-histidine peptide, such as the tag provided in a pQEvector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311),among others, many of which are commercially available. As described inGentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), forinstance, hexa-histidine provides for convenient purification of thefusion protein. Another peptide tag useful for purification, the “HA”tag, corresponds to an epitope derived from the influenza hemagglutininprotein. (Wilson et al., Cell 37:767 (1984).)

[0184] Thus, any of these above fusions can be engineered using thepolynucleotides or the polypeptides of the present invention.

[0185] Nucleic acids encoding the above epitopes can also be recombinedwith a gene of interest as an epitope tag (e.g., the hemagglutinin(“HA”) tag or flag tag) to aid in detection and purification of theexpressed polypeptide. For example, a system described by Janknecht etal. allows for the ready purification of non-denatured fusion proteinsexpressed in human cell lines (Janknecht et al., Proc. Natl. Acad. Sci.USA 88:8972-897 (1991)). In this system, the gene of interest issubcloned into a vaccinia recombination plasmid such that the openreading frame of the gene is translationally fused to an amino-terminaltag consisting of six histidine residues. The tag serves as a matrixbinding domain for the fusion protein. Extracts from cells infected withthe recombinant vaccinia virus are loaded onto Ni2+ nitriloaceticacid-agarose column and histidine-tagged proteins can be selectivelyeluted with imidazole-containing buffers.

[0186] Additional fusion proteins of the invention may be generatedthrough the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”). DNA shuffling may be employed to modulate the activities ofpolypeptides of the invention, such methods can be used to generatepolypeptides with altered activity, as well as agonists and antagonistsof the polypeptides. See, generally, U.S. Pat. Nos. 5,605,793;5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr.Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol.16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999);and Lorenzo and Blasco, Biotechniques 24(2):308-13 (1998) (each of thesepatents and publications are hereby incorporated by reference in itsentirety). In one embodiment, alteration of polynucleotidescorresponding to SEQ ID NO:X and the polypeptides encoded by thesepolynucleotides may be achieved by DNA shuffling. DNA shuffling involvesthe assembly of two or more DNA segments by homologous or site-specificrecombination to generate variation in the polynucleotide sequence. Inanother embodiment, polynucleotides of the invention, or the encodedpolypeptides, may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of a polynucleotide encodinga polypeptide of the invention may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules.

[0187] Polynucleotide and Polypeptide Variants

[0188] The invention also encompasses apoptosis related variants. Thepresent invention is directed to variants of the polynucleotide sequencedisclosed in SEQ ID NO:X or the complementary strand thereto, and/or thecDNA sequence contained in cDNA plasmid:Z.

[0189] The present invention also encompasses variants of thepolypeptide sequence disclosed in SEQ ID NO:Y, a polypeptide sequenceencoded by the polynucleotide sequence in SEQ ID NO:X and/or apolypeptide sequence encoded by the cDNA in cDNA plasmid:Z.

[0190] “Variant” refers to a polynucleotide or polypeptide differingfrom the polynucleotide or polypeptide of the present invention, butretaining properties thereof. Generally, variants are overall closelysimilar, and, in many regions, identical to the polynucleotide orpolypeptide of the present invention.

[0191] Thus, one aspect of the invention provides an isolated nucleicacid molecule comprising, or alternatively consisting of, apolynucleotide having a nucleotide sequence selected from the groupconsisting of: (a) a nucleotide sequence encoding a apoptosis relatedpolypeptide having an amino acid sequence as shown in the sequencelisting and described in SEQ ID NO:X or the cDNA in cDNA plasmid:Z; (b)a nucleotide sequence encoding a mature apoptosis related polypeptidehaving the amino acid sequence as shown in the sequence listing anddescribed in SEQ ID NO:X or the cDNA in cDNA plasmid:Z; (c) a nucleotidesequence encoding a biologically active fragment of a apoptosis relatedpolypeptide having an amino acid sequence shown in the sequence listingand described in SEQ ID NO:X or the cDNA in cDNA plasmid:Z; (d) anucleotide sequence encoding an antigenic fragment of a apoptosisrelated polypeptide having an amino acid sequence shown in the sequencelisting and described in SEQ ID NO:X or the cDNA in cDNA plasmid:Z; (e)a nucleotide sequence encoding a apoptosis related polypeptidecomprising the complete amino acid sequence encoded by a human cDNAplasmid contained in SEQ ID NO:X or the cDNA in cDNA plasmid:Z; (f) anucleotide sequence encoding a mature apoptosis related polypeptidehaving an amino acid sequence encoded by a human cDNA plasmid containedin SEQ ID NO:X or the cDNA in cDNA plasmid:Z; (g) a nucleotide sequenceencoding a biologically active fragment of a apoptosis relatedpolypeptide having an amino acid sequence encoded by a human cDNAplasmid contained in SEQ ID NO:X or the cDNA in cDNA plasmid:Z; (h) anucleotide sequence encoding an antigenic fragment of a apoptosisrelated polypeptide having an amino acid sequence encoded by a humancDNA plasmid contained in SEQ ID NO:X or the cDNA in cDNA plasmid:Z; (i)a nucleotide sequence complementary to any of the nucleotide sequencesin (a), (b), (c), (d), (e), (f), (g), or (h), above.

[0192] The present invention is also directed to nucleic acid moleculeswhich comprise, or alternatively consist of, a nucleotide sequence whichis at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identicalto, for example, any of the nucleotide sequences in (a), (b), (c), (d),(e), (f), (g), (h), or (i) above. Polypeptides encoded by these nucleicacid molecules are also encompassed by the invention. In anotherembodiment, the invention encompasses nucleic acid molecules whichcomprise, or alternatively, consist of a polynucleotide which hybridizesunder stringent hybridization conditions, or alternatively, under lowerstringency conditions, to a polynucleotide in (a), (b), (c), (d), (e),(f), (g), (h), or (i), above. Polynucleotides which hybridize to thecomplement of these nucleic acid molecules under stringent hybridizationconditions or alternatively, under lower stringency conditions, are alsoencompassed by the invention, as are polypeptides encoded by thesepolynucleotides.

[0193] Another aspect of the invention provides an isolated nucleic acidmolecule comprising, or alternatively consisting of, a polynucleotidehaving a nucleotide sequence selected from the group consisting of: (a)a nucleotide sequence encoding a apoptosis related polypeptide having anamino acid sequence as shown in the sequence listing and described inTable 1; (b) a nucleotide sequence encoding a mature apoptosis relatedpolypeptide having the amino acid sequence as shown in the sequencelisting and described in Table 1; (c) a nucleotide sequence encoding abiologically active fragment of a apoptosis related polypeptide havingan amino acid sequence shown in the sequence listing and described inTable 1; (d) a nucleotide sequence encoding an antigenic fragment of aapoptosis related polypeptide having an amino acid sequence shown in thesequence listing and described in Table 1; (e) a nucleotide sequenceencoding a apoptosis related polypeptide comprising the complete aminoacid sequence encoded by a human cDNA in a cDNA plasmid contained in theATCC Deposit and described in Table 1; (f) a nucleotide sequenceencoding a mature apoptosis related polypeptide having an amino acidsequence encoded by a human cDNA in a cDNA plasmid contained in the ATCCDeposit and described in Table 1; (g) a nucleotide sequence encoding abiologically active fragment of a apoptosis related polypeptide havingan amino acid sequence encoded by a human cDNA in a cDNA plasmidcontained in the ATCC Deposit and described in Table 1; (h) a nucleotidesequence encoding an antigenic fragment of a apoptosis relatedpolypeptide having an amino acid sequence encoded by a human cDNA in acDNA plasmid contained in the ATCC Deposit and described in Table 1; (i)a nucleotide sequence complementary to any of the nucleotide sequencesin (a), (b), (c), (d), (e), (f), (g), or (h), above.

[0194] The present invention is also directed to nucleic acid moleculeswhich comprise, or alternatively consist of, a nucleotide sequence whichis at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identicalto, for example, any of the nucleotide sequences in (a), (b), (c), (d),(e), (f), (g), (h), or (i) above. Polypeptides encoded by these nucleicacid molecules are also encompassed by the invention. In anotherembodiment, the invention encompasses nucleic acid molecules whichcomprise, or alternatively, consist of a polynucleotide which hybridizesunder stringent hybridization conditions, or alternatively, under lowerstringency conditions, to a polynucleotide in (a), (b), (c), (d), (e),(f), (g), (h), or (i), above. Polynucleotides which hybridize to thecomplement of these nucleic acid molecules under stringent hybridizationconditions or alternatively, under lower stringency conditions, are alsoencompassed by the invention, as are polypeptides encoded by thesepolynucleotides.

[0195] The present invention is also directed to polypeptides whichcomprise, or alternatively consist of, an amino acid sequence which isat least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to,for example, the polypeptide sequence shown in SEQ ID NO:Y, apolypeptide sequence encoded by the nucleotide sequence in SEQ ID NO:X,a polypeptide sequence encoded by the cDNA in cDNA plasmid:Z, and/orpolypeptide fragments of any of these polypeptides (e.g., thosefragments described herein). Polynucleotides which hybridize to thecomplement of the nucleic acid molecules encoding these polypeptidesunder stringent hybridization conditions or alternatively, under lowerstringency conditions are also encompassed by the invention, as arepolypeptides encoded by these polynucleotides.

[0196] By a nucleic acid having a nucleotide sequence at least, forexample, 95% “identical” to a reference nucleotide sequence of thepresent invention, it is intended that the nucleotide sequence of thenucleic acid is identical to the reference sequence except that thenucleotide sequence may include up to five point mutations per each 100nucleotides of the reference nucleotide sequence encoding thepolypeptide. In other words, to obtain a nucleic acid having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. The query sequence may bean entire sequence referred to in Table 1, the ORF (open reading frame),or any fragment specified as described herein.

[0197] As a practical matter, whether any particular nucleic acidmolecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% identical to a nucleotide sequence of the present invention can bedetermined conventionally using known computer programs. A preferredmethod for determining the best overall match between a query sequence(a sequence of the present invention) and a subject sequence, alsoreferred to as a global sequence alignment, can be determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. 6:237-245 (1990)). In a sequence alignment the query andsubject sequences are both DNA sequences. An RNA sequence can becompared by converting U's to T's. The result of said global sequencealignment is in percent identity. Preferred parameters used in a FASTDBalignment of DNA sequences to calculate percent identify are:Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty 0.05, Window Size=500 or the lenght of the subject nucleotidesequence, whichever is shorter.

[0198] If the subject sequence is shorter than the query sequencebecause of 5′ or 3′ deletions, not because of internal deletions, amanual correction must be made to the results. This is because theFASTDB program does not account for 5′ and 3′ truncations of the subjectsequence when calculating percent identity. For subject sequencestruncated at the 5′ or 3′ ends, relative to the query sequence, thepercent identity is corrected by calculating the number of bases of thequery sequence that are 5′ and 3′ of the subject sequence, which are notmatched/aligned, as a percent of the total bases of the query sequence.Whether a nucleotide is matched/aligned is determined by results of theFASTDB sequence alignment. This percentage is then subtracted from thepercent identity, calculated by the above FASTDB program using thespecified parameters, to arrive at a final percent identity score. Thiscorrected score is what is used for the purposes of the presentinvention. Only bases outside the 5′ and 3′ bases of the subjectsequence, as displayed by the FASTDB alignment, which are notmatched/aligned with the query sequence, are calculated for the purposesof manually adjusting the percent identity score.

[0199] For example, a 90 base subject sequence is aligned to a 100 basequery sequence to determine percent identity. The deletions occur at the5′ end of the subject sequence and therefore, the FASTDB alignment doesnot show a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

[0200] By a polypeptide having an amino acid sequence at least, forexample, 95% “identical” to a query amino acid sequence of the presentinvention, it is intended that the amino acid sequence of the subjectpolypeptide is identical to the query sequence except that the subjectpolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the query amino acid sequence. In other words,to obtain a polypeptide having an amino acid sequence at least 95%identical to a query amino acid sequence, up to 5% of the amino acidresidues in the subject sequence may be inserted, deleted, (indels) orsubstituted with another amino acid. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

[0201] As a practical matter, whether any particular polypeptide is atleast 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, forinstance, the amino acid sequence referred to in Table 1 or a fragmentthereof, the amino acid sequence encoded by the nucleotide sequence inSEQ ID NO:X or a fragment thereof, or to the amino acid sequence encodedby the cDNA in cDNA plasmid:Z, or a fragment thereof, can be determinedconventionally using known computer programs. A preferred method fordetermining the best overall match between a query sequence (a sequenceof the present invention) and a subject sequence, also referred to as aglobal sequence alignment, can be determined using the FASTDB computerprogram based on the algorithm of Brutlag et al. (Comp. App.Biosci.6:237-245(1990)). In a sequence alignment the query and subjectsequences are either both nucleotide sequences or both amino acidsequences. The result of said global sequence alignment is in percentidentity. Preferred parameters used in a FASTDB amino acid alignmentare: Matrix=PAM 0, k-tuple-2, Mismatch Penalty=1, Joining Penalty=20,Randomization Group Length=0, Cutoff Score=l, Window Size=sequencelength, Gap Penalty=5, Gap Size Penalty-0.05, Window Size=500 or thelength of the subject amino acid sequence, whichever is shorter.

[0202] If the subject sequence is shorter than the query sequence due toN- or C-terminal deletions, not because of internal deletions, a manualcorrection must be made to the results. This is because the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N- and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of the presentinvention. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence.

[0203] For example, a 90 amino acid residue subject sequence is alignedwith a 100 residue query sequence to determine percent identity. Thedeletion occurs at the N-terminus of the subject sequence and therefore,the FASTDB alignment does not show a matching/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only residue positions outside the N-and C-terminal ends of the subject sequence, as displayed in the FASTDBalignment, which are not matched/aligned with the query sequnce aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

[0204] The variants may contain alterations in the coding regions,non-coding regions, or both. Especially preferred are polynucleotidevariants containing alterations which produce silent substitutions,additions, or deletions, but do not alter the properties or activitiesof the encoded polypeptide. Nucleotide variants produced by silentsubstitutions due to the degeneracy of the genetic code are preferred.Moreover, variants in which less than 50, less than 40, less than 30,less than 20, less than 10, or 5-50, 5-25, 5-10, 1-5, or 1-2 amino acidsare substituted, deleted, or added in any combination are alsopreferred. Polynucleotide variants can be produced for a variety ofreasons, e.g., to optimize codon expression for a particular host(change codons in the human mRNA to those preferred by a bacterial hostsuch as E. coli).

[0205] Naturally occurring variants are called “allelic variants,” andrefer to one of several alternate forms of a gene occupying a givenlocus on a chromosome of an organism. (Genes II, Lewin, B., ed., JohnWiley & Sons, New York (1985).) These allelic variants can vary ateither the polynucleotide and/or polypeptide level and are included inthe present invention. Alternatively, non-naturally occurring variantsmay be produced by mutagenesis techniques or by direct synthesis.

[0206] Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the polypeptides of the present invention. Forinstance, as discussed herein, one or more amino acids can be deletedfrom the N-terminus or C-terminus of the polypeptide of the presentinvention without substantial loss of biological function. The authorsof Ron et al., J. Biol. Chem. 268: 2984-2988 (1993), reported variantKGF proteins having heparin binding activity even after deleting 3, 8,or 27 amino-terminal amino acid residues. Similarly, Interferon gammaexhibited up to ten times higher activity after deleting 8-10 amino acidresidues from the carboxy terminus of this protein. (Dobeli et al., J.Biotechnology 7:199-216 (1988).)

[0207] Moreover, ample evidence demonstrates that variants often retaina biological activity similar to that of the naturally occurringprotein. For example, Gayle and coworkers (J. Biol. Chem 268:22105-22111(1993)) conducted extensive mutational analysis of human cytokine IL-1a.They used random mutagenesis to generate over 3,500 individual IL-1amutants that averaged 2.5 amino acid changes per variant over the entirelength of the molecule. Multiple mutations were examined at everypossible amino acid position. The investigators found that “[m]ost ofthe molecule could be altered with little effect on either [binding orbiological activity].” (See, Abstract.) In fact, only 23 unique aminoacid sequences, out of more than 3,500 nucleotide sequences examined,produced a protein that significantly differed in activity fromwild-type.

[0208] Furthermore, as discussed herein, even if deleting one or moreamino acids from the N-terminus or C-terminus of a polypeptide resultsin modification or loss of one or more biological functions, otherbiological activities may still be retained. For example, the ability ofa deletion variant to induce and/or to bind antibodies which recognizethe secreted form will likely be retained when less than the majority ofthe residues of the secreted form are removed from the N-terminus orC-terminus. Whether a particular polypeptide lacking N- or C-terminalresidues of a protein retains such immunogenic activities can readily bedetermined by routine methods described herein and otherwise known inthe art.

[0209] Thus, the invention further includes polypeptide variants whichshow a functional activity (e.g. biological activity) of the polypeptideof the invention, of which they are a variant. Such variants includedeletions, insertions, inversions, repeats, and substitutions selectedaccording to general rules known in the art so as have little effect onactivity.

[0210] The present application is directed to nucleic acid molecules atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to thenucleic acid sequences disclosed herein, (e.g., encoding a polypeptidehaving the amino acid sequence of an N and/or C terminal deletion),irrespective of whether they encode a polypeptide having functionalactivity. This is because even where a particular nucleic acid moleculedoes not encode a polypeptide having functional activity, one of skillin the art would still know how to use the nucleic acid molecule, forinstance, as a hybridization probe or a polymerase chain reaction (PCR)primer. Uses of the nucleic acid molecules of the present invention thatdo not encode a polypeptide having functional activity include, interalia, (1) isolating a gene or allelic or splice variants thereof in acDNA library; (2) in situ hybridization (e.g., “FISH”) to metaphasechromosomal spreads to provide precise chromosomal location of the gene,as described in Verma et al., Human Chromosomes: A Manual of BasicTechniques, Pergamon Press, New York (1988); and (3) Northern Blotanalysis for detecting mRNA expression in specific tissues.

[0211] Preferred, however, are nucleic acid molecules having sequencesat least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to thenucleic acid sequences disclosed herein, which do, in fact, encode apolypeptide having functional activity of a polypeptide of theinvention.

[0212] Of course, due to the degeneracy of the genetic code, one ofordinary skill in the art will immediately recognize that a large numberof the nucleic acid molecules having a sequence at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100% identical to, for example, the nucleicacid sequence of the cDNA in cDNA plasmid:Z, the nucleic acid sequencereferred to in Table 1 (SEQ ID NO:X), or fragments thereof, will encodepolypeptides “having functional activity.” In fact, since degeneratevariants of any of these nucleotide sequences all encode the samepolypeptide, in many instances, this will be clear to the skilledartisan even without performing the above described comparison assay. Itwill be further recognized in the art that, for such nucleic acidmolecules that are not degenerate variants, a reasonable number willalso encode a polypeptide having functional activity. This is becausethe skilled artisan is fully aware of amino acid substitutions that areeither less likely or not likely to significantly effect proteinfunction (e.g., replacing one aliphatic amino acid with a secondaliphatic amino acid), as further described below.

[0213] For example, guidance concerning how to make phenotypicallysilent amino acid substitutions is provided in Bowie et al.,“Deciphering the Message in Protein Sequences: Tolerance to Amino AcidSubstitutions,” Science 247:1306-1310 (1990), wherein the authorsindicate that there are two main strategies for studying the toleranceof an amino acid sequence to change.

[0214] The first strategy exploits the tolerance of amino acidsubstitutions by natural selection during the process of evolution. Bycomparing amino acid sequences in different species, conserved aminoacids can be identified. These conserved amino acids are likelyimportant for protein function. In contrast, the amino acid positionswhere substitutions have been tolerated by natural selection indicatesthat these positions are not critical for protein function. Thus,positions tolerating amino acid substitution could be modified whilestill maintaining biological activity of the protein.

[0215] The second strategy uses genetic engineering to introduce aminoacid changes at specific positions of a cloned gene to identify regionscritical for protein function. For example, site directed mutagenesis oralanine-scanning mutagenesis (introduction of single alanine mutationsat every residue in the molecule) can be used. (Cunningham and Wells,Science 244:1081-1085 (1989).) The resulting mutant molecules can thenbe tested for biological activity.

[0216] As the authors state, these two strategies have revealed thatproteins are surprisingly tolerant of amino acid substitutions. Theauthors further indicate which amino acid changes are likely to bepermissive at certain amino acid positions in the protein. For example,most buried (within the tertiary structure of the protein) amino acidresidues require nonpolar side chains, whereas few features of surfaceside chains are generally conserved. Moreover, tolerated conservativeamino acid substitutions involve replacement of the aliphatic orhydrophobic amino acids Ala, Val, Leu and Ile; replacement of thehydroxyl residues Ser and Thr; replacement of the acidic residues Aspand Glu; replacement of the amide residues Asn and Gln, replacement ofthe basic residues Lys, Arg, and His; replacement of the aromaticresidues Phe, Tyr, and Trp, and replacement of the small-sized aminoacids Ala, Ser, Thr, Met, and Gly. Besides conservative amino acidsubstitution, variants of the present invention include (i)substitutions with one or more of the non-conserved amino acid residues,where the substituted amino acid residues may or may not be one encodedby the genetic code, or (ii) substitution with one or more of amino acidresidues having a substituent group, or (iii) fusion of the maturepolypeptide with another compound, such as a compound to increase thestability and/or solubility of the polypeptide (for example,polyethylene glycol), or (iv) fusion of the polypeptide with additionalamino acids, such as, for example, an IgG Fc fusion region peptide, orleader or secretory sequence, or a sequence facilitating purification or(v) fusion of the polypeptide with another compound, such as albumin(including but not limited to recombinant albumin (see, e.g., U.S. Pat.No. 5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat.No. 5,766,883, issued Jun. 16, 1998, herein incorporated by reference intheir entirety)). Such variant polypeptides are deemed to be within thescope of those skilled in the art from the teachings herein.

[0217] For example, polypeptide variants containing amino acidsubstitutions of charged amino acids with other charged or neutral aminoacids may produce proteins with improved characteristics, such as lessaggregation. Aggregation of pharmaceutical formulations both reducesactivity and increases clearance due to the aggregate's immunogenicactivity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).)

[0218] A further embodiment of the invention relates to a polypeptidewhich comprises the amino acid sequence of a polypeptide having an aminoacid sequence which contains at least one amino acid substitution, butnot more than 50 amino acid substitutions, even more preferably, notmore than 40 amino acid substitutions, still more preferably, not morethan 30 amino acid substitutions, and still even more preferably, notmore than 20 amino acid substitutions. Of course it is highly preferablefor a polypeptide to have an amino acid sequence which comprises theamino acid sequence of a polypeptide of SEQ ID NO:Y, an amino acidsequence encoded by SEQ ID NO:X, and/or the amino acid sequence encodedby the cDNA in cDNA plasmid:Z which contains, in order ofever-increasing preference, at least one, but not more than 10, 9, 8, 7,6, 5, 4, 3, 2 or 1 amino acid substitutions. In specific embodiments,the number of additions, substitutions, and/or deletions in the aminoacid sequence of SEQ ID NO:Y or fragments thereof (e.g., the mature formand/or other fragments described herein), an amino acid sequence encodedby SEQ ID NO:X or fragments thereof, and/or the amino acid sequenceencoded by cDNA plasmid:Z or fragments thereof, is 1-5, 5-10, 5-25,5-50, 10-50 or 50-150, conservative amino acid substitutions arepreferable. As discussed herein, any polypeptide of the presentinvention can be used to generate fusion proteins. For example, thepolypeptide of the present invention, when fused to a second protein,can be used as an antigenic tag. Antibodies raised against thepolypeptide of the present invention can be used to indirectly detectthe second protein by binding to the polypeptide. Moreover, becausesecreted proteins target cellular locations based on traffickingsignals, polypeptides of the present invention which are shown to besecreted can be used as targeting molecules once fused to otherproteins.

[0219] Examples of domains that can be fused to polypeptides of thepresent invention include not only heterologous signal sequences, butalso other heterologous functional regions. The fusion does notnecessarily need to be direct, but may occur through linker sequences.

[0220] In certain preferred embodiments, proteins of the inventioncomprise fusion proteins wherein the polypeptides are N and/orC-terminal deletion mutants. In preferred embodiments, the applicationis directed to nucleic acid molecules at least 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% identical to the nucleic acid sequences encodingpolypeptides having the amino acid sequence of the specific N- andC-terminal deletions mutants. Polynucleotides encoding thesepolypeptides, including fragments and/or variants, are also encompassedby the invention.

[0221] Moreover, fusion proteins may also be engineered to improvecharacteristics of the polypeptide of the present invention. Forinstance, a region of additional amino acids, particularly charged aminoacids, may be added to the N-terminus of the polypeptide to improvestability and persistence during purification from the host cell orsubsequent handling and storage. Also, peptide moieties may be added tothe polypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to facilitate handling of polypeptides are familiar and routinetechniques in the art.

[0222] As one of skill in the art will appreciate, polypeptides of thepresent invention of the present invention and the epitope-bearingfragments thereof described above can be combined with heterologouspolypeptide sequences. For example, the polypeptides of the presentinvention may be fused with heterologous polypeptide sequences, forexample, the polypeptides of the present invention may be fused with theconstant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portionsthereof (CH1, CH2, CH3, and any combination thereof, including bothentire domains and portions thereof), resulting in chimericpolypeptides. These fusion proteins facilitate purification and show anincreased half-life in vivo. One reported example describes chimericproteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. (EP A 394,827; Trauneckeret al., Nature 331:84-86 (1988).) Fusion proteins havingdisulfide-linked dimeric structures (due to the IgG) can also be moreefficient in binding and neutralizing other molecules, than themonomeric protein or protein fragment alone. (Fountoulakis et al., J.Biochem. 270:3958-3964 (1995).)

[0223] Vectors, Host Cells, and Protein Production

[0224] The present invention also relates to vectors containing thepolynucleotide of the present invention, host cells, and the productionof polypeptides by recombinant techniques. The vector may be, forexample, a phage, plasmid, viral, or retroviral vector. Retroviralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally will occur only incomplementing host cells.

[0225] The polynucleotides of the invention may be joined to a vectorcontaining a selectable marker for propagation in a host. Generally, aplasmid vector is introduced in a precipitate, such as a calciumphosphate precipitate, or in a complex with a charged lipid. If thevector is a virus, it may be packaged in vitro using an appropriatepackaging cell line and then transduced into host cells.

[0226] The polynucleotide insert should be operatively linked to anappropriate promoter, such as the phage lambda PL promoter, the E. colilac, trp, phoA and tac promoters, the SV40 early and late promoters andpromoters of retroviral LTRs, to name a few. Other suitable promoterswill be known to the skilled artisan. The expression constructs willfurther contain sites for transcription initiation, termination, and, inthe transcribed region, a ribosome binding site for translation. Thecoding portion of the transcripts expressed by the constructs willpreferably include a translation initiating codon at the beginning and atermination codon (UAA, UGA or UAG) appropriately positioned at the endof the polypeptide to be translated.

[0227] As indicated, the expression vectors will preferably include atleast one selectable marker. Such markers include dihydrofolatereductase, G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Representative examples of appropriate hostsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCCAccession No. 201178)); insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowesmelanoma cells; and plant cells. Appropriate culture mediums andconditions for the above-described host cells are known in the art.

[0228] Among vectors preferred for use in bacteria include pQE70, pQE60and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescriptvectors, pNH8A, pNH16a, pNH18A, pNH46A, available from StratageneCloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia Biotech, Inc. Among preferred eukaryoticvectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available fromStratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.Preferred expression vectors for use in yeast systems include, but arenot limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ,pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, andPAO815 (all available from Invitrogen, Carlbad, Calif.). Other suitablevectors will be readily apparent to the skilled artisan.

[0229] Introduction of the construct into the host cell can be effectedby calcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection, or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986). It is specifically contemplated that the polypeptides ofthe present invention may in fact be expressed by a host cell lacking arecombinant vector.

[0230] A polypeptide of this invention can be recovered and purifiedfrom recombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.

[0231] Polypeptides of the present invention can also be recovered from:products purified from natural sources, including bodily fluids, tissuesand cells, whether directly isolated or cultured; products of chemicalsynthetic procedures; and products produced by recombinant techniquesfrom a prokaryotic or eukaryotic host, including, for example,bacterial, yeast, higher plant, insect, and mammalian cells. Dependingupon the host employed in a recombinant production procedure, thepolypeptides of the present invention may be glycosylated or may benon-glycosylated. In addition, polypeptides of the invention may alsoinclude an initial modified methionine residue, in some cases as aresult of host-mediated processes. Thus, it is well known in the artthat the N-terminal methionine encoded by the translation initiationcodon generally is removed with high efficiency from any protein aftertranslation in all eukaryotic cells. While the N-terminal methionine onmost proteins also is efficiently removed in most prokaryotes, for someproteins, this prokaryotic removal process is inefficient, depending onthe nature of the amino acid to which the N-terminal methionine iscovalently linked.

[0232] In one embodiment, the yeast Pichia pastoris is used to expresspolypeptides of the invention in a eukaryotic system. Pichia pastoris isa methylotrophic yeast which can metabolize methanol as its sole carbonsource. A main step in the methanol metabolization pathway is theoxidation of methanol to formaldehyde using O₂. This reaction iscatalyzed by the enzyme alcohol oxidase. In order to metabolize methanolas its sole carbon source, Pichia pastoris must generate high levels ofalcohol oxidase due, in part, to the relatively low affinity of alcoholoxidase for O₂. Consequently, in a growth medium depending on methanolas a main carbon source, the promoter region of one of the two alcoholoxidase genes (AOX1) is highly active. In the presence of methanol,alcohol oxidase produced from the AOX1 gene comprises up toapproximately 30% of the total soluble protein in Pichia pastoris. See,Ellis, S. B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P. J, etal., Yeast 5:167-77 (1989); Tschopp, J. F., et al., Nucl. Acids Res.15:3859-76 (1987). Thus, a heterologous coding sequence, such as, forexample, a polynucleotide of the present invention, under thetranscriptional regulation of all or part of the AOX1 regulatorysequence is expressed at exceptionally high levels in Pichia yeast grownin the presence of methanol.

[0233] In one example, the plasmid vector pPIC9K is used to express DNAencoding a polypeptide of the invention, as set forth herein, in aPichea yeast system essentially as described in “Pichia Protocols:Methods in Molecular Biology,” D. R. Higgins and J. Cregg, eds. TheHumana Press, Totowa, N.J., 1998. This expression vector allowsexpression and secretion of a polypeptide of the invention by virtue ofthe strong AOX1 promoter linked to the Pichia pastoris alkalinephosphatase (PHO) secretory signal peptide (i.e., leader) locatedupstream of a multiple cloning site.

[0234] Many other yeast vectors could be used in place of pPIC9K, suchas, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9,pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815, as one skilled in theart would readily appreciate, as long as the proposed expressionconstruct provides appropriately located signals for transcription,translation, secretion (if desired), and the like, including an in-frameAUG as required.

[0235] In another embodiment, high-level expression of a heterologouscoding sequence, such as, for example, a polynucleotide of the presentinvention, may be achieved by cloning the heterologous polynucleotide ofthe invention into an expression vector such as, for example, pGAPZ orpGAPZalpha, and growing the yeast culture in the absence of methanol.

[0236] In addition to encompassing host cells containing the vectorconstructs discussed herein, the invention also encompasses primary,secondary, and immortalized host cells of vertebrate origin,particularly mammalian origin, that have been engineered to delete orreplace endogenous genetic material (e.g., coding sequence), and/or toinclude genetic material (e.g., heterologous polynucleotide sequences)that is operably associated with polynucleotides of the invention, andwhich activates, alters, and/or amplifies endogenous polynucleotides.For example, techniques known in the art may be used to operablyassociate heterologous control regions (e.g., promoter and/or enhancer)and endogenous polynucleotide sequences via homologous recombination(see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; InternationalPublication No. WO 96/29411, published Sep. 26, 1996; InternationalPublication No. WO 94/12650, published Aug. 4, 1994; Koller et al.,Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al.,Nature 342:435-438 (1989), the disclosures of each of which areincorporated by reference in their entireties).

[0237] In addition, polypeptides of the invention can be chemicallysynthesized using techniques known in the art (e.g., see Creighton,1983, Proteins: Structures and Molecular Principles, W. H. Freeman &Co., N.Y., and Hunkapiller et al., Nature, 310:105-111 (1984)). Forexample, a polypeptide corresponding to a fragment of a polypeptide canbe synthesized by use of a peptide synthesizer. Furthermore, if desired,nonclassical amino acids or chemical amino acid analogs can beintroduced as a substitution or addition into the polypeptide sequence.Non-classical amino acids include, but are not limited to, to theD-isomers of the common amino acids, 2,4-diaminobutyric acid, a-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu,e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline,sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine,t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine,fluoro-amino acids, designer amino acids such as b-methyl amino acids,Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs ingeneral. Furthermore, the amino acid can be D (dextrorotary) or L(levorotary).

[0238] The invention encompasses polypeptides of the present inventionwhich are differentially modified during or after translation, e.g., byglycosylation, acetylation, phosphorylation, amidation, derivatizationby known protecting/blocking groups, proteolytic cleavage, linkage to anantibody molecule or other cellular ligand, etc. Any of numerouschemical modifications may be carried out by known techniques, includingbut not limited, to specific chemical cleavage by cyanogen bromide,trypsin, chymotrypsin, papain, V8 protease, NaBH₄; acetylation,formylation, oxidation, reduction; metabolic synthesis in the presenceof tunicamycin; etc.

[0239] Additional post-translational modifications encompassed by theinvention include, for example, e.g., N-linked or O-linked carbohydratechains, processing of N-terminal or C-terminal ends), attachment ofchemical moieties to the amino acid backbone, chemical modifications ofN-linked or O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein.

[0240] Also provided by the invention are chemically modifiedderivatives of the polypeptides of the invention which may provideadditional advantages such as increased solubility, stability andcirculating time of the polypeptide, or decreased immunogenicity (seeU.S. Pat. No. 4,179,337). The chemical moieties for derivitization maybe selected from water soluble polymers such as polyethylene glycol,ethylene glycol/propylene glycol copolymers, carboxymethylcellulose,dextran, polyvinyl alcohol and the like. The polypeptides may bemodified at random positions within the molecule, or at predeterminedpositions within the molecule and may include one, two, three or moreattached chemical moieties.

[0241] The polymer may be of any molecular weight, and may be branchedor unbranched. For polyethylene glycol, the preferred molecular weightis between about 1 kDa and about 100 kDa (the term “about” indicatingthat in preparations of polyethylene glycol, some molecules will weighmore, some less, than the stated molecular weight) for ease in handlingand manufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

[0242] The polyethylene glycol molecules (or other chemical moieties)should be attached to the protein with consideration of effects onfunctional or antigenic domains of the protein. There are a number ofattachment methods available to those skilled in the art, e.g., EP 0 401384, herein incorporated by reference (coupling PEG to G-CSF), see alsoMalik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue. Sulfhydryl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecules. Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group.

[0243] One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (polypeptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

[0244] The polypeptides of the invention may be in monomers or multimers(i.e., dimers, trimers, tetramers and higher multimers). Accordingly,the present invention relates to monomers and multimers of thepolypeptides of the invention, their preparation, and compositions(preferably, Therapeutics) containing them. In specific embodiments, thepolypeptides of the invention are monomers, dimers, trimers ortetramers. In additional embodiments, the multimers of the invention areat least dimers, at least trimers, or at least tetramers.

[0245] Multimers encompassed by the invention may be homomers orheteromers. As used herein, the term homomer, refers to a multimercontaining only polypeptides corresponding to the amino acid sequence ofSEQ ID NO:Y or an amino acid sequence encoded by SEQ ID NO:X or thecomplement of SEQ ID NO:X, and/or an amino acid sequence encoded by cDNAplasmid:Z (including fragments, variants, splice variants, and fusionproteins, corresponding to these as described herein). These homomersmay contain polypeptides having identical or different amino acidsequences. In a specific embodiment, a homomer of the invention is amultimer containing only polypeptides having an identical amino acidsequence. In another specific embodiment, a homomer of the invention isa multimer containing polypeptides having different amino acidsequences. In specific embodiments, the multimer of the invention is ahomodimer (e.g., containing polypeptides having identical or differentamino acid sequences) or a homotrimer (e.g., containing polypeptideshaving identical and/or different amino acid sequences). In additionalembodiments, the homomeric multimer of the invention is at least ahomodimer, at least a homotrimer, or at least a homotetramer.

[0246] As used herein, the term heteromer refers to a multimercontaining one or more heterologous polypeptides (i.e., polypeptides ofdifferent proteins) in addition to the polypeptides of the invention. Ina specific embodiment, the multimer of the invention is a heterodimer, aheterotrimer, or a heterotetramer. In additional embodiments, theheteromeric multimer of the invention is at least a heterodimer, atleast a heterotrimer, or at least a heterotetramer.

[0247] Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked, by for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when polypeptides of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when polypeptides of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentassociations with and/or between the polypeptides of the invention. Suchcovalent associations may involve one or more amino acid residuescontained in the polypeptide sequence (e.g., that recited in SEQ IDNO:Y, or contained in a polypeptide encoded by SEQ ID NO:X, and/or thecDNA plasmid:Z). In one instance, the covalent associations arecross-linking between cysteine residues located within the polypeptidesequences which interact in the native (i.e., naturally occurring)polypeptide. In another instance, the covalent associations are theconsequence of chemical or recombinant manipulation. Alternatively, suchcovalent associations may involve one or more amino acid residuescontained in the heterologous polypeptide sequence in a fusion protein.In one example, covalent associations are between the heterologoussequence contained in a fusion protein of the invention (see, e.g., U.S.Pat. No. 5,478,925). In a specific example, the covalent associationsare between the heterologous sequence contained in a Fc fusion proteinof the invention (as described herein). In another specific example,covalent associations of fusion proteins of the invention are betweenheterologous polypeptide sequence from another protein that is capableof forming covalently associated multimers, such as for example,osteoprotegerin (see, e.g., International Publication NO: WO 98/49305,the contents of which are herein incorporated by reference in itsentirety). In another embodiment, two or more polypeptides of theinvention are joined through peptide linkers. Examples include thosepeptide linkers described in U.S. Pat. No. 5,073,627 (herebyincorporated by reference). Proteins comprising multiple polypeptides ofthe invention separated by peptide linkers may be produced usingconventional recombinant DNA technology.

[0248] Another method for preparing multimer polypeptides of theinvention involves use of polypeptides of the invention fused to aleucine zipper or isoleucine zipper polypeptide sequence. Leucine zipperand isoleucine zipper domains are polypeptides that promotemultimerization of the proteins in which they are found. Leucine zipperswere originally identified in several DNA-binding proteins (Landschulzet al., Science 240:1759, (1988)), and have since been found in avariety of different proteins. Among the known leucine zippers arenaturally occurring peptides and derivatives thereof that dimerize ortrimerize. Examples of leucine zipper domains suitable for producingsoluble multimeric proteins of the invention are those described in PCTapplication WO 94/10308, hereby incorporated by reference. Recombinantfusion proteins comprising a polypeptide of the invention fused to apolypeptide sequence that dimerizes or trimerizes in solution areexpressed in suitable host cells, and the resulting soluble multimericfusion protein is recovered from the culture supernatant usingtechniques known in the art.

[0249] Trimeric polypeptides of the invention may offer the advantage ofenhanced biological activity. Preferred leucine zipper moieties andisoleucine moieties are those that preferentially form trimers. Oneexample is a leucine zipper derived from lung surfactant protein D(SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) andin U.S. patent application Ser. No. 08/446,922, hereby incorporated byreference. Other peptides derived from naturally occurring trimericproteins may be employed in preparing trimeric polypeptides of theinvention.

[0250] In another example, proteins of the invention are associated byinteractions between Flag® polypeptide sequence contained in fusionproteins of the invention containing Flag® polypeptide seuqence. In afurther embodiment, associations proteins of the invention areassociated by interactions between heterologous polypeptide sequencecontained in Flag® fusion proteins of the invention and anti-Flag®antibody.

[0251] The multimers of the invention may be generated using chemicaltechniques known in the art. For example, polypeptides desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the sequence ofthe polypeptides desired to be contained in the multimer (see, e.g.,U.S. Pat. No. 5,478,925, which is herein incorporated by reference inits entirety). Further, polypeptides of the invention may be routinelymodified by the addition of cysteine or biotin to the C-terminus orN-terminus of the polypeptide and techniques known in the art may beapplied to generate multimers containing one or more of these modifiedpolypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety). Additionally, techniquesknown in the art may be applied to generate liposomes containing thepolypeptide components desired to be contained in the multimer of theinvention (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety).

[0252] Alternatively, multimers of the invention may be generated usinggenetic engineering techniques known in the art. In one embodiment,polypeptides contained in multimers of the invention are producedrecombinantly using fusion protein technology described herein orotherwise known in the art (see, e.g., U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety). In a specificembodiment, polynucleotides coding for a homodimer of the invention aregenerated by ligating a polynucleotide sequence encoding a polypeptideof the invention to a sequence encoding a linker polypeptide and thenfurther to a synthetic polynucleotide encoding the translated product ofthe polypeptide in the reverse orientation from the original C-terminusto the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat.No. 5,478,925, which is herein incorporated by reference in itsentirety). In another embodiment, recombinant techniques describedherein or otherwise known in the art are applied to generate recombinantpolypeptides of the invention which contain a transmembrane domain (orhyrophobic or signal peptide) and which can be incorporated by membranereconstitution techniques into liposomes (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).

[0253] Antibodies

[0254] Further polypeptides of the invention relate to antibodies andT-cell antigen receptors (TCR) which immunospecifically bind apolypeptide, polypeptide fragment, or variant of SEQ iID NO:Y, and/or anepitope, of the present invention (as determined by immunoassays wellknown in the art for assaying specific antibody-antigen binding).Antibodies of the invention include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized or chimeric antibodies,single chain antibodies, Fab fragments, F(ab′) fragments, fragmentsproduced by a Fab expression library, anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies of theinvention), and epitope-binding fragments of any of the above. The term“antibody,” as used herein, refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds an antigen. The immunoglobulin molecules of the invention can beof any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

[0255] Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdfv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine (e.g., mouse andrat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.As used herein, “human” antibodies include antibodies having the aminoacid sequence of a human immunoglobulin and include antibodies isolatedfrom human immunoglobulin libraries or from animals transgenic for oneor more human immunoglobulin and that do not express endogenousimmunoglobulins, as described infra and, for example in, U.S. Pat. No.5,939,598 by Kucherlapati et al.

[0256] The antibodies of the present invention may be monospecific,bispecific, trispecific or of greater multispecificity. Multispecificantibodies may be specific for different epitopes of a polypeptide ofthe present invention or may be specific for both a polypeptide of thepresent invention as well as for a heterologous epitope, such as aheterologous polypeptide or solid support material. See, e.g., PCTpublications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt,et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893;4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

[0257] Antibodies of the present invention may be described or specifiedin terms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, or by size in contiguous amino acidresidues. Antibodies which specifically bind any epitope or polypeptideof the present invention may also be excluded. Therefore, the presentinvention includes antibodies that specifically bind polypeptides of thepresent invention, and allows for the exclusion of the same.

[0258] Antibodies of the present invention may also be described orspecified in terms of their cross-reactivity. Antibodies that do notbind any other analog, ortholog, or homolog of a polypeptide of thepresent invention are included. Antibodies that bind polypeptides withat least 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55%, and at least 50%identity (as calculated using methods known in the art and describedherein) to a polypeptide of the present invention are also included inthe present invention. In specific embodiments, antibodies of thepresent invention cross-react with murine, rat and/or rabbit homologs ofhuman proteins and the corresponding epitopes thereof. Antibodies thatdo not bind polypeptides with less than 95%, less than 90%, less than85%, less than 80%, less than 75%, less than 70%, less than 65%, lessthan 60%, less than 55%, and less than 50% identity (as calculated usingmethods known in the art and described herein) to a polypeptide of thepresent invention are also included in the present invention. In aspecific embodiment, the above-described cross-reactivity is withrespect to any single specific antigenic or immunogenic polypeptide, orcombination(s) of 2, 3, 4, 5, or more of the specific antigenic and/orimmunogenic polypeptides disclosed herein. Further included in thepresent invention are antibodies which bind polypeptides encoded bypolynucleotides which hybridize to a polynucleotide of the presentinvention under stringent hybridization conditions (as describedherein). Antibodies of the present invention may also be described orspecified in terms of their binding affinity to a polypeptide of theinvention. Preferred binding affinities include those with adissociation constant or Kd less than 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶M, 5×10⁻⁷ M, 10⁷ M,5×10⁻⁸M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹M, 5×10⁻¹² M, ¹⁰⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵M, or 10−¹⁵ M.

[0259] The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least95%, at least 90%, at least 85 %, at least 80%, at least 75%, at least70%, at least 60%, or at least 50%.

[0260] Antibodies of the present invention may act as agonists orantagonists of the polypeptides of the present invention. For example,the present invention includes antibodies which disrupt thereceptor/ligand interactions with the polypeptides of the inventioneither partially or fully. Preferrably, antibodies of the presentinvention bind an antigenic epitope disclosed herein, or a portionthereof. The invention features both receptor-specific antibodies andligand-specific antibodies. The invention also featuresreceptor-specific antibodies which do not prevent ligand binding butprevent receptor activation. Receptor activation (i.e., signaling) maybe determined by techniques described herein or otherwise known in theart. For example, receptor activation can be determined by detecting thephosphorylation (e.g., tyrosine or serine/threonine) of the receptor orits substrate by immunoprecipitation followed by western blot analysis(for example, as described supra). In specific embodiments, antibodiesare provided that inhibit ligand activity or receptor activity by atleast 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 60%, or at least 50% of the activity in absence ofthe antibody.

[0261] The invention also features receptor-specific antibodies whichboth prevent ligand binding and receptor activation as well asantibodies that recognize the receptor-ligand complex, and, preferably,do not specifically recognize the unbound receptor or the unboundligand. Likewise, included in the invention are neutralizing antibodieswhich bind the ligand and prevent binding of the ligand to the receptor,as well as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies which activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation, for example, byinducing dimerization of the receptor. The antibodies may be specifiedas agonists, antagonists or inverse agonists for biological activitiescomprising the specific biological activities of the peptides of theinvention disclosed herein. The above antibody agonists can be madeusing methods known in the art. See, e.g., PCT publication WO 96/40281;U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chenet al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol.161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214(1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al.,J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J. Immunol.Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241(1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997);Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(l):14-20 (1996)(which are all incorporated by reference herein in their entireties).

[0262] Antibodies of the present invention may be used, for example, butnot limited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides of the present invention in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988) (incorporated by reference hereinin its entirety).

[0263] As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, radionuclides, or toxins. See, e.g.,PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 396,387.

[0264] The antibodies of the invention include derivatives that aremodified, i.e, by the covalent attachment of any type of molecule to theantibody such that covalent attachment does not prevent the antibodyfrom generating an anti-idiotypic response. For example, but not by wayof limitation, the antibody derivatives include antibodies that havebeen modified, e.g., by glycosylation, acetylation, pegylation,phosphylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

[0265] The antibodies of the present invention may be generated by anysuitable method known in the art. Polyclonal antibodies to anantigen-of-interest can be produced by various procedures well known inthe art. For example, a polypeptide of the invention can be administeredto various host animals including, but not limited to, rabbits, mice,rats, etc. to induce the production of sera containing polyclonalantibodies specific for the antigen. Various adjuvants may be used toincrease the immunological response, depending on the host species, andinclude but are not limited to, Freund's (complete and incomplete),mineral gels such as aluminum hydroxide, surface active substances suchas lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants are also well known in the art.

[0266] Monoclonal antibodies can be prepared using a wide variety oftechniques known in the art including the use of hybridoma, recombinant,and phage display technologies, or a combination thereof. For example,monoclonal antibodies can be produced using hybridoma techniquesincluding those known in the art and taught, for example, in Harlow etal., Antibodies: A Laboratory Manual, (Cold Spring Harbor LaboratoryPress, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies andT-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said referencesincorporated by reference in their entireties). The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. The term “monoclonal antibody” refers to anantibody that is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.

[0267] Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art and arediscussed in detail in the Examples. In a non-limiting example, mice canbe immunized with a polypeptide of the invention or a cell expressingsuch peptide. Once an immune response is detected, e.g., antibodiesspecific for the antigen are detected in the mouse serum, the mousespleen is harvested and splenocytes isolated. The splenocytes are thenfused by well known techniques to any suitable myeloma cells, forexample cells from cell line SP20 available from the ATCC. Hybridomasare selected and cloned by limited dilution. The hybridoma clones arethen assayed by methods known in the art for cells that secreteantibodies capable of binding a polypeptide of the invention. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by immunizing mice with positive hybridoma clones.

[0268] Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

[0269] Antibody fragments which recognize specific epitopes may begenerated by known techniques. For example, Fab and F(ab′)2 fragments ofthe invention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

[0270] For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies of the present invention includethose disclosed in Brinkman et al., J. Immunol. Methods 182:41-50(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al.,Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280(1994); PCT application No. PCT/GB91/01134; PCT publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

[0271] As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties).

[0272] Examples of techniques which can be used to produce single-chainFvs and antibodies include those described in U.S. Pat. Nos. 4,946,778and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991);Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science240:1038-1040 (1988) For some uses, including in vivo use of antibodiesin humans and in vitro detection assays, it may be preferable to usechimeric, humanized, or human antibodies. A chimeric antibody is amolecule in which different portions of the antibody are derived fromdifferent animal species, such as antibodies having a variable regionderived from a murine monoclonal antibody and a human immunoglobulinconstant region. Methods for producing chimeric antibodies are known inthe art. See e.g., Morrison, Science 229:1202 (1985); Oi et al.,BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816397, whichare incorporated herein by reference in their entirety. Humanizedantibodies are antibody molecules from non-human species antibody thatbinds the desired antigen having one or more complementarity determiningregions (CDRs) from the non-human species and a framework regions from ahuman immunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. (See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., Nature 332:323 (1988), which areincorporated herein by reference in their entireties.) Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332).

[0273] Completely human antibodies are particularly desirable fortherapeutic treatment of human patients. Human antibodies can be made bya variety of methods known in the art including phage display methodsdescribed above using antibody libraries derived from humanimmunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893,WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which isincorporated herein by reference in its entirety.

[0274] Human antibodies can also be produced using transgenic mice whichare incapable of expressing functional endogenous immunoglobulins, butwhich can express human immunoglobulin genes. For example, the humanheavy and light chain immunoglobulin gene complexes may be introducedrandomly or by homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homologous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homologousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;5,885,793; 5,916,771; and 5,939,598, which are incorporated by referenceherein in their entirety. In addition, companies such as Abgenix, Inc.(Freemont, Calif.) and Genpharm (San Jose, Calif.) can be engaged toprovide human antibodies directed against a selected antigen usingtechnology similar to that described above.

[0275] Completely human antibodies which recognize a selected epitopecan be generated using a technique referred to as “guided selection.” Inthis approach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

[0276] Further, antibodies to the polypeptides of the invention can, inturn, be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444;(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

[0277] Polynucleotides Encoding Antibodies

[0278] The invention farther provides polynucleotides comprising anucleotide sequence encoding an antibody of the invention and fragmentsthereof. The invention also encompasses polynucleotides that hybridizeunder stringent or alternatively, under lower stringency hybridizationconditions, e.g., as defined supra, to polynucleotides that encode anantibody, preferably, that specifically binds to a polypeptide of theinvention, preferably, an antibody that binds to a polypeptide havingthe amino acid sequence of SEQ ID NO:Y.

[0279] The polynucleotides may be obtained, and the nucleotide sequenceof the polynucleotides determined, by any method known in the art. Forexample, if the nucleotide sequence of the antibody is known, apolynucleotide encoding the antibody may be assembled from chemicallysynthesized oligonucleotides (e.g., as described in Kutmeier et al.,BioTechniques 17:242 (1994)), which, briefly, involves the synthesis ofoverlapping oligonucleotides containing portions of the sequenceencoding the antibody, annealing and ligating of those oligonucleotides,and then amplification of the ligated oligonucleotides by PCR.

[0280] Alternatively, a polynucleotide encoding an antibody may begenerated from nucleic acid from a suitable source. If a clonecontaining a nucleic acid encoding a particular antibody is notavailable, but the sequence of the antibody molecule is known, a nucleicacid encoding the immunoglobulin may be chemically synthesized orobtained from a suitable source (e.g., an antibody cDNA library, or acDNA library generated from, or nucleic acid, preferably poly A+ RNA,isolated from, any tissue or cells expressing the antibody, such ashybridoma cells selected to express an antibody of the invention) by PCRamplification using synthetic primers hybridizable to the 3′ and 5′ endsof the sequence or by cloning using an oligonucleotide probe specificfor the particular gene sequence to identify, e.g., a cDNA clone from acDNA library that encodes the antibody. Amplified nucleic acidsgenerated by PCR may then be cloned into replicable cloning vectorsusing any method well known in the art.

[0281] Once the nucleotide sequence and corresponding amino acidsequence of the antibody is determined, the nucleotide sequence of theantibody may be manipulated using methods well known in the art for themanipulation of nucleotide sequences, e.g., recombinant DNA techniques,site directed mutagenesis, PCR, etc. (see, for example, the techniquesdescribed in Sambrook et al., 1990, Molecular Cloning, A LaboratoryManual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology,John Wiley & Sons, NY, which are both incorporated by reference hereinin their entireties ), to generate antibodies having a different aminoacid sequence, for example to create amino acid substitutions,deletions, and/or insertions.

[0282] In a specific embodiment, the amino acid sequence of the heavyand/or light chain variable domains may be inspected to identify thesequences of the complementarity determining regions (CDRs) by methodsthat are well know in the art, e.g., by comparison to known amino acidsequences of other heavy and light chain variable regions to determinethe regions of sequence hypervariability. Using routine recombinant DNAtechniques, one or more of the CDRs may be inserted within frameworkregions, e.g., into human framework regions to humanize a non-humanantibody, as described supra. The framework regions may be naturallyoccurring or consensus framework regions, and preferably human frameworkregions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998)for a listing of human framework regions). Preferably, thepolynucleotide generated by the combination of the framework regions andCDRs encodes an antibody that specifically binds a polypeptide of theinvention. Preferably, as discussed supra, one or more amino acidsubstitutions may be made within the framework regions, and, preferably,the amino acid substitutions improve binding of the antibody to itsantigen. Additionally, such methods may be used to make amino acidsubstitutions or deletions of one or more variable region cysteineresidues participating in an intrachain disulfide bond to generateantibody molecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

[0283] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

[0284] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42(1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988);and Ward et al., Nature 334:544-54 (1989)) can be adapted to producesingle chain antibodies. Single chain antibodies are formed by linkingthe heavy and light chain fragments of the Fv region via an amino acidbridge, resulting in a single chain polypeptide. Techniques for theassembly of functional Fv fragments in E. coli may also be used (Skerraet al., Science 242:1038-1041 (1988)).

[0285] Methods of Producing Antibodies

[0286] The antibodies of the invention can be produced by any methodknown in the art for the synthesis of antibodies, in particular, bychemical synthesis or preferably, by recombinant expression techniques.

[0287] Recombinant expression of an antibody of the invention, orfragment, derivative or analog thereof, (e.g., a heavy or light chain ofan antibody of the invention or a single chain antibody of theinvention), requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

[0288] The expression vector is transferred to a host cell byconventional techniques and the transfected cells are then cultured byconventional techniques to produce an antibody of the invention. Thus,the invention includes host cells containing a polynucleotide encodingan antibody of the invention, or a heavy or light chain thereof, or asingle chain antibody of the invention, operably linked to aheterologous promoter. In preferred embodiments for the expression ofdouble-chained antibodies, vectors encoding both the heavy and lightchains may be co-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

[0289] A variety of host-expression vector systems may be utilized toexpress the antibody molecules of the invention. Such host-expressionsystems represent vehicles by which the coding sequences of interest maybe produced and subsequently purified, but also represent cells whichmay, when transformed or transfected with the appropriate nucleotidecoding sequences, express an antibody molecule of the invention in situ.These include but are not limited to microorganisms such as bacteria(e.g., E. coli, B. subtilis) transformed with recombinant bacteriophageDNA, plasmid DNA or cosmid DNA expression vectors containing antibodycoding sequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

[0290] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

[0291] In an insect system, Autographa californica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperda cells. The antibody coding sequence maybe cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

[0292] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the antibody coding sequence of interest may beligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericgene may then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing the antibody molecule in infectedhosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359(1984)). Specific initiation signals may also be required for efficienttranslation of inserted antibody coding sequences. These signals includethe ATG initiation codon and adjacent sequences. Furthermore, theinitiation codon must be in phase with the reading frame of the desiredcoding sequence to ensure translation of the entire insert. Theseexogenous translational control signals and initiation codons can be ofa variety of origins, both natural and synthetic. The efficiency ofexpression may be enhanced by the inclusion of appropriate transcriptionenhancer elements, transcription terminators, etc. (see Bittner et al.,Methods in Enzymol. 153:51-544 (1987)).

[0293] In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, W138, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

[0294] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress the antibody molecule may be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

[0295] A number of selection systems may be used, including but notlimited to the herpes simplex virus thymidine kinase (Wigler et al.,Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), andadenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980))genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).Methods commonly known in the art of recombinant DNA technology may beroutinely applied to select the desired recombinant clone, and suchmethods are described, for example, in Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), CurrentProtocols in Human Genetics, John Wiley & Sons, NY (1994);Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which areincorporated by reference herein in their entireties.

[0296] The expression levels of an antibody molecule can be increased byvector amplification (for a review, see Bebbington and Hentschel, Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol.3. (Academic Press, NewYork, 1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

[0297] The host cell may be co-transfected with two expression vectorsof the invention, the first vector encoding a heavy chain derivedpolypeptide and the second vector encoding a light chain derivedpolypeptide. The two vectors may contain identical selectable markerswhich enable equal expression of heavy and light chain polypeptides.Alternatively, a single vector may be used which encodes, and is capableof expressing, both heavy and light chain polypeptides. In suchsituations, the light chain should be placed before the heavy chain toavoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52(1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The codingsequences for the heavy and light chains may comprise cDNA or genomicDNA.

[0298] Once an antibody molecule of the invention has been produced byan animal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

[0299] The present invention encompasses antibodies recombinantly fusedor chemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of thepolypeptide) of the present invention to generate fusion proteins. Thefusion does not necessarily need to be direct, but may occur throughlinker sequences. The antibodies may be specific for antigens other thanpolypeptides (or portion thereof, preferably at least 10, 20, 30, 40,50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the presentinvention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol.146:2446-2452(1991), which are incorporated by reference in theirentireties.

[0300] The present invention further includes compositions comprisingthe polypeptides of the present invention fused or conjugated toantibody domains other than the variable regions. For example, thepolypeptides of the present invention may be fused or conjugated to anantibody Fc region, or portion thereof. The antibody portion fused to apolypeptide of the present invention may comprise the constant region,hinge region, CH1 domain, CH2 domain, and CH3 domain or any combinationof whole domains or portions thereof. The polypeptides may also be fusedor conjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol.154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA89:11337-11341(1992) (said references incorporated by reference in theirentireties).

[0301] As discussed, supra, the polypeptides corresponding to apolypeptide, polypeptide fragment, or a variant of SEQ ID NO:Y may befused or conjugated to the above antibody portions to increase the invivo half life of the polypeptides or for use in immunoassays usingmethods known in the art. Further, the polypeptides corresponding to SEQID NO:Y may be fused or conjugated to the above antibody portions tofacilitate purification. One reported example describes chimericproteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. (EP 394,827; Traunecker etal., Nature 331:84-86 (1988). The polypeptides of the present inventionfused or conjugated to an antibody having disulfide-linked dimericstructures (due to the IgG) may also be more efficient in binding andneutralizing other molecules, than the monomeric secreted protein orprotein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964(1995)). In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP A 232,262). Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,Bennett et al., J. Molecular Recognition 8:52-58 (1995); Johanson etal., J. Biol. Chem. 270:9459-9471 (1995).

[0302] Moreover, the antibodies or fragments thereof of the presentinvention can be fused to marker sequences, such as a peptide tofacilitate purification. In preferred embodiments, the marker amino acidsequence is a hexa-histidine peptide, such as the tag provided in a pQEvector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311),among others, many of which are commercially available. As described inGentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), forinstance, hexa-histidine provides for convenient purification of thefusion protein. Other peptide tags useful for purification include, butare not limited to, the “HA” tag, which corresponds to an epitopederived from the influenza hemagglutinin protein (Wilson et al., Cell37:767 (1984)) and the “flag” tag.

[0303] The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to theantibody (or fragment thereof) or indirectly, through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art. See, for example, U.S. Pat. No. 4,741,900 for metal ionswhich can be conjugated to antibodies for use as diagnostics accordingto the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude 125I, 131I, 111In or 99Tc.

[0304] Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells. Examples includepaclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthrarnycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

[0305] The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, a-interferon, 1-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See,International Publication No. WO 97/33899), AIM II (See, InternationalPublication No. WO 97134911), Fas Ligand (Takahashi et al., Int.Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No.WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

[0306] Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

[0307] Techniques for conjugating such therapeutic moiety to antibodiesare well known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

[0308] Alternatively, an antibody can be conjugated to a second antibodyto form an antibody heteroconjugate as described by Segal in U.S. Pat.No. 4,676,980, which is incorporated herein by reference in itsentirety.

[0309] An antibody, with or without a therapeutic moiety conjugated toit, administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

[0310] Immunophenotyping

[0311] The antibodies of the invention may be utilized forimmunophenotyping of cell lines and biological samples. The translationproduct of the gene of the present invention may be useful as a cellspecific marker, or more specifically as a cellular marker that isdifferentially expressed at various stages of differentiation and/ormaturation of particular cell types. Monoclonal antibodies directedagainst a specific epitope, or combination of epitopes, will allow forthe screening of cellular populations expressing the marker. Varioustechniques can be utilized using monoclonal antibodies to screen forcellular populations expressing the marker(s), and include magneticseparation using antibody-coated magnetic beads, “panning” with antibodyattached to a solid matrix (i.e., plate), and flow cytometry (See, e.g.,U.S. Pat. No. 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

[0312] These techniques allow for the screening of particularpopulations of cells, such as might be found with hematologicalmalignancies (i.e. minimal residual disease (MRD) in acute leukemicpatients) and “non-self” cells in transplantations to preventGraft-versus-Host Disease (GVHD). Alternatively, these techniques allowfor the screening of hematopoietic stem and progenitor cells capable ofundergoing proliferation and/or differentiation, as might be found inhuman umbilical cord blood.

[0313] Assays for Antibody Binding

[0314] The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimnmunoassays, protein A immunoassays, to name but a few. Such assaysare routine and well known in the art (see, e.g., Ausubel et al, eds,1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,Inc., New York, which is incorporated by reference herein in itsentirety). Exemplary immunoassays are described briefly below (but arenot intended by way of limitation).

[0315] Immunoprecipitation protocols generally comprise lysing apopulation of cells in a lysis buffer such as RIPA buffer (1% NP-40 orTriton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 Msodium phosphate at pH 7.2, 1% Trasylol) supplemented with proteinphosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin,sodium vanadate), adding the antibody of interest to the cell lysate,incubating for a period of time (e.g., 1-4 hours) at 4° C., addingprotein A and/or protein G sepharose beads to the cell lysate,incubating for about an hour or more at 4° C., washing the beads inlysis buffer and resuspending the beads in SDS/sample buffer. Theability of the antibody of interest to immunoprecipitate a particularantigen can be assessed by, e.g., western blot analysis. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the binding of the antibody to an antigen and decrease thebackground (e.g., pre-clearing the cell lysate with sepharose beads).For further discussion regarding immunoprecipitation protocols see,e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology,Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

[0316] Western blot analysis generally comprises preparing proteinsamples, electrophoresis of the protein samples in a polyacrylamide gel(e.g., 8%-20% SDS-PAGE depending on the molecular weight of theantigen), transferring the protein sample from the polyacrylamide gel toa membrane such as nitrocellulose, PVDF or nylon, blocking the membranein blocking solution (e.g., PBS with 3% BSA or non-fat milk), washingthe membrane in washing buffer (e.g., PBS-Tween 20), blocking themembrane with primary antibody (the antibody of interest) diluted inblocking buffer, washing the membrane in washing buffer, blocking themembrane with a secondary antibody (which recognizes the primaryantibody, e.g., an anti-human antibody) conjugated to an enzymaticsubstrate (e.g., horseradish peroxidase or alkaline phosphatase) orradioactive molecule (e.g., 32P or 125I) diluted in blocking buffer,washing the membrane in wash buffer, and detecting the presence of theantigen. One of skill in the art would be knowledgeable as to theparameters that can be modified to increase the signal detected and toreduce the background noise. For further discussion regarding westernblot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols inMolecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

[0317] ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

[0318] The binding affinity of an antibody to an antigen and theoff-rate of an antibody-antigen interaction can be determined bycompetitive binding assays. One example of a competitive binding assayis a radioimmunoassay comprising the incubation of labeled antigen(e.g., 3H or 125I) with the antibody of interest in the presence ofincreasing amounts of unlabeled antigen, and the detection of theantibody bound to the labeled antigen. The affinity of the antibody ofinterest for a particular antigen and the binding off-rates can bedetermined from the data by scatchard plot analysis. Competition with asecond antibody can also be determined using radioimmunoassays. In thiscase, the antigen is incubated with antibody of interest conjugated to alabeled compound (e.g., 3H or 125I) in the presence of increasingamounts of an unlabeled second antibody.

[0319] Therapeutic Uses

[0320] The present invention is further directed to antibody-basedtherapies which involve administering antibodies of the invention to ananimal, preferably a mammal, and most preferably a human, patient fortreating one or more of the disclosed diseases, disorders, orconditions. Therapeutic compounds of the invention include, but are notlimited to, antibodies of the invention (including fragments, analogsand derivatives thereof as described herein) and nucleic acids encodingantibodies of the invention (including fragments, analogs andderivatives thereof and anti-idiotypic antibodies as described herein).The antibodies of the invention can be used to treat, inhibit or preventdiseases, disorders or conditions associated with aberrant expressionand/or activity of a polypeptide of the invention, including, but notlimited to, any one or more of the diseases, disorders, or conditionsdescribed herein. The treatment and/or prevention of diseases,disorders, or conditions associated with aberrant expression and/oractivity of a polypeptide of the invention includes, but is not limitedto, alleviating symptoms associated with those diseases, disorders orconditions. Antibodies of the invention may be provided inpharmaceutically acceptable compositions as known in the art or asdescribed herein.

[0321] A summary of the ways in which the antibodies of the presentinvention may be used therapeutically includes binding polynucleotidesor polypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

[0322] The antibodies of this invention may be advantageously utilizedin combination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

[0323] The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, humanantibodies, fragments derivatives, analogs, or nucleic acids, areadministered to a human patient for therapy or prophylaxis.

[0324] It is preferred to use high affinity and/or potent in vivoinhibiting and/or neutralizing antibodies against polypeptides orpolynucleotides of the present invention, fragments or regions thereof,for both immunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides of theinvention, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10⁻² M,10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M,10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M,10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10¹³ M, 10⁻¹³ M,5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, and 10⁻¹⁵ M.

[0325] Gene Therapy

[0326] In a specific embodiment, nucleic acids comprising sequencesencoding antibodies or functional derivatives thereof, are administeredto treat, inhibit or prevent a disease or disorder associated withaberrant expression and/or activity of a polypeptide of the invention,by way of gene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

[0327] Any of the methods for gene therapy available in the art can beused according to the present invention. Exemplary methods are describedbelow.

[0328] For general reviews of the methods of gene therapy, see Goldspielet al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596(1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson,Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH 11 (5):155-215(1993). Methods commonly known in the art of recombinant DNA technologywhich can be used are described in Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, NY (1993); andKriegler, Gene Transfer and Expression, A Laboratory Manual, StocktonPress, NY (1990).

[0329] In a preferred aspect, the compound comprises nucleic acidsequences encoding an antibody, said nucleic acid sequences being partof expression vectors that express the antibody or fragments or chimericproteins or heavy or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue-specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody encodingnucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). Inspecific embodiments, the expressed antibody molecule is a single chainantibody; alternatively, the nucleic acid sequences include sequencesencoding both the heavy and light chains, or fragments thereof, of theantibody.

[0330] Delivery of the nucleic acids into a patient may be eitherdirect, in which case the patient is directly exposed to the nucleicacid or nucleic acid-carrying vectors, or indirect, in which case, cellsare first transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

[0331] In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987))(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635;WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller and Smithies, Proc.Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature342:435-438 (1989)).

[0332] In a specific embodiment, viral vectors that contains nucleicacid sequences encoding an antibody of the invention are used. Forexample, a retroviral vector can be used (see Miller et al., Meth.Enzymol. 217:581-599 (1993)). These retroviral vectors contain thecomponents necessary for the correct packaging of the viral genome andintegration into the host cell DNA. The nucleic acid sequences encodingthe antibody to be used in gene therapy are cloned into one or morevectors, which facilitates delivery of the gene into a patient. Moredetail about retroviral vectors can be found in Boesen et al.,Biotherapy 6:291-302 (1994), which describes the use of a retroviralvector to deliver the mdr1 gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy are: Cloweset al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141(1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel.3:110-114 (1993).

[0333] Adenoviruses are other viral vectors that can be used in genetherapy. Adenoviruses are especially attractive vehicles for deliveringgenes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In apreferred embodiment, adenovirus vectors are used.

[0334] Adeno-associated virus (AAV) has also been proposed for use ingene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300(1993); U.S. Pat. No. 5,436,146).

[0335] Another approach to gene therapy involves transferring a gene tocells in tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

[0336] In this embodiment, the nucleic acid is introduced into a cellprior to administration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993);Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

[0337] The resulting recombinant cells can be delivered to a patient byvarious methods known in the art. Recombinant blood cells (e.g.,hematopoietic stem or progenitor cells) are preferably administeredintravenously. The amount of cells envisioned for use depends on thedesired effect, patient state, etc., and can be determined by oneskilled in the art.

[0338] Cells into which a nucleic acid can be introduced for purposes ofgene therapy encompass any desired, available cell type, and include butare not limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

[0339] In a preferred embodiment, the cell used for gene therapy isautologous to the patient.

[0340] In an embodiment in which recombinant cells are used in genetherapy, nucleic acid sequences encoding an antibody are introduced intothe cells such that they are expressible by the cells or their progeny,and the recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see e.g. PCT Publication WO 94/08598; Stemple andAnderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229(1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

[0341] In a specific embodiment, the nucleic acid to be introduced forpurposes of gene therapy comprises an inducible promoter operably linkedto the coding region, such that expression of the nucleic acid iscontrollable by controlling the presence or absence of the appropriateinducer of transcription. Demonstration of Therapeutic or ProphylacticActivity.

[0342] The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

[0343] Therapeutic/Prophylactic Administration and Composition

[0344] The invention provides methods of treatment, inhibition andprophylaxis by administration to a subject of an effective amount of acompound or pharmaceutical composition of the invention, preferably apolypeptide or antibody of the invention. In a preferred aspect, thecompound is substantially purified (e.g., substantially free fromsubstances that limit its effect or produce undesired side-effects). Thesubject is preferably an animal, including but not limited to animalssuch as cows, pigs, horses, chickens, cats, dogs, etc., and ispreferably a mammal, and most preferably human.

[0345] Formulations and methods of administration that can be employedwhen the compound comprises a nucleic acid or an immunoglobulin aredescribed above; additional appropriate formulations and routes ofadministration can be selected from among those described herein below.

[0346] Various delivery systems are known and can be used to administera compound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds or compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compounds or compositions of the invention into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

[0347] In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

[0348] In another embodiment, the compound or composition can bedelivered in a vesicle, in particular a liposome (see Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; seegenerally ibid.)

[0349] In yet another embodiment, the compound or composition can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201(1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl.J. Med. 321:574 (1989)). In another embodiment, polymeric materials canbe used (see Medical Applications of Controlled Release, Langer and Wise(eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci.Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190(1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al.,J.Neurosurg. 71:105 (1989)). In yet another embodiment, a controlledrelease system can be placed in proximity of the therapeutic target,i.e., the brain, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol.2, pp.115-138 (1984)).

[0350] Other controlled release systems are discussed in the review byLanger (Science 249:1527-1533 (1990)).

[0351] In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

[0352] The present invention also provides pharmaceutical compositions.Such compositions comprise a therapeutically effective amount of acompound, and a pharmaceutically acceptable carrier. In a specificembodiment, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

[0353] In a preferred embodiment, the composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic such as lignocaineto ease pain at the site of the injection. Generally, the ingredientsare supplied either separately or mixed together in unit dosage form,for example, as a dry lyophilized powder or water free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration.

[0354] The compounds of the invention can be formulated as neutral orsalt forms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

[0355] The amount of the compound of the invention which will beeffective in the treatment, inhibition and prevention of a disease ordisorder associated with aberrant expression and/or activity of apolypeptide of the invention can be determined by standard clinicaltechniques. In addition, in vitro assays may optionally be employed tohelp identify optimal dosage ranges. The precise dose to be employed inthe formulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

[0356] For antibodies, the dosage administered to a patient is typically0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, thedosage administered to a patient is between 0.1 mg/kg and 20 mg/kg ofthe patient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

[0357] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

[0358] Diagnosis and Imaging

[0359] Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases, disorders,and/or conditions associated with the aberrant expression and/oractivity of a polypeptide of the invention. The invention provides forthe detection of aberrant expression of a polypeptide of interest,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of aberrant expression.

[0360] The invention provides a diagnostic assay for diagnosing adisorder, comprising (a) assaying the expression of the polypeptide ofinterest in cells or body fluid of an individual using one or moreantibodies specific to the polypeptide interest and (b) comparing thelevel of gene expression with a standard gene expression level, wherebyan increase or decrease in the assayed polypeptide gene expression levelcompared to the standard expression level is indicative of a particulardisorder. With respect to cancer, the presence of a relatively highamount of transcript in biopsied tissue from an individual may indicatea predisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0361] Antibodies of the invention can be used to assay protein levelsin a biological sample using classical immunohistological methods knownto those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, et al., J. Cell . Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

[0362] One aspect of the invention is the detection and diagnosis of adisease or disorder associated with aberrant expression of a polypeptideof interest in an animal, preferably a mammal and most preferably ahuman. In one embodiment, diagnosis comprises: a) administering (forexample, parenterally, subcutaneously, or intraperitoneally) to asubject an effective amount of a labeled molecule which specificallybinds to the polypeptide of interest; b) waiting for a time intervalfollowing the administering for permitting the labeled molecule topreferentially concentrate at sites in the subject where the polypeptideis expressed (and for unbound labeled molecule to be cleared tobackground level); c) determining background level; and d) detecting thelabeled molecule in the subject, such that detection of labeled moleculeabove the background level indicates that the subject has a particulardisease or disorder associated with aberrant expression of thepolypeptide of interest. Background level can be determined by variousmethods including, comparing the amount of labeled molecule detected toa standard value previously determined for a particular system.

[0363] It will be understood in the art that the size of the subject andthe imaging system used will determine the quantity of imaging moietyneeded to produce diagnostic images. In the case of a radioisotopemoiety, for a human subject, the quantity of radioactivity injected willnormally range from about 5 to 20 millicuries of 99mTc. The labeledantibody or antibody fragment will then preferentially accumulate at thelocation of cells which contain the specific protein. In vivo tumorimaging is described in S. W. Burchiel et al., “lmmunopharnacokineticsof Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982).

[0364] Depending on several variables, including the type of label usedand the mode of administration, the time interval following theadministration for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject and for unbound labeled molecule tobe cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to12 hours. In another embodiment the time interval followingadministration is 5 to 20 days or 5 to 10 days.

[0365] In an embodiment, monitoring of the disease or disorder iscarried out by repeating the method for diagnosing the disease ordisease, for example, one month after initial diagnosis, six monthsafter initial diagnosis, one year after initial diagnosis, etc.

[0366] Presence of the labeled molecule can be detected in the patientusing methods known in the art for in vivo scanning. These methodsdepend upon the type of label used. Skilled artisans will be able todetermine the appropriate method for detecting a particular label.Methods and devices that may be used in the diagnostic methods of theinvention include, but are not limited to, computed tomography (CT),whole body scan such as position emission tomography (PET), magneticresonance imaging (MRI), and sonography.

[0367] In a specific embodiment, the molecule is labeled with aradioisotope and is detected in the patient using a radiation responsivesurgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). Inanother embodiment, the molecule is labeled with a fluorescent compoundand is detected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

[0368] Kits

[0369] The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

[0370] In another specific embodiment of the present invention, the kitis a diagnostic kit for use in screening serum containing antibodiesspecific against proliferative and/or cancerous polynucleotides andpolypeptides. Such a kit may include a control antibody that does notreact with the polypeptide of interest. Such a kit may include asubstantially isolated polypeptide antigen comprising an epitope whichis specifically immunoreactive with at least one anti-polypeptideantigen antibody. Further, such a kit includes means for detecting thebinding of said antibody to the antigen (e.g., the antibody may beconjugated to a fluorescent compound such as fluorescein or rhodaminewhich can be detected by flow cytometry). In specific embodiments, thekit may include a recombinantly produced or chemically synthesizedpolypeptide antigen. The polypeptide antigen of the kit may also beattached to a solid support.

[0371] In a more specific embodiment the detecting means of theabove-described kit includes a solid support to which said polypeptideantigen is attached. Such a kit may also include a non-attachedreporter-labeled anti-human antibody. In this embodiment, binding of theantibody to the polypeptide antigen can be detected by binding of thesaid reporter-labeled antibody.

[0372] In an additional embodiment, the invention includes a diagnostickit for use in screening serum containing antigens of the polypeptide ofthe invention. The diagnostic kit includes a substantially isolatedantibody specifically immunoreactive with polypeptide or polynucleotideantigens, and means for detecting the binding of the polynucleotide orpolypeptide antigen to the antibody. In one embodiment, the antibody isattached to a solid support. In a specific embodiment, the antibody maybe a monoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

[0373] In one diagnostic configuration, test serum is reacted with asolid phase reagent having a surface-bound antigen obtained by themethods of the present invention. After binding with specific antigenantibody to the reagent and removing unbound serum components bywashing, the reagent is reacted with reporter-labeled anti-humanantibody to bind reporter to the reagent in proportion to the amount ofbound anti-antigen antibody on the solid support. The reagent is againwashed to remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or calorimetric substrate(Sigmna, St. Louis, Mo.).

[0374] The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

[0375] Thus, the invention provides an assay system or kit for carryingout this diagnostic method. The kit generally includes a support withsurface- bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

[0376] Uses of the Polynucleotides

[0377] Each of the polynucleotides identified herein can be used innumerous ways as reagents. The following description should beconsidered exemplary and utilizes known techniques.

[0378] The polynucleotides of the present invention are useful forchromosome identification. There exists an ongoing need to identify newchromosome markers, since few chromosome marking reagents, based onactual sequence data (repeat polymorphisms), are presently available.Each sequence is specifically targeted to and can hybridize with aparticular location on an individual human chromosome, thus eachpolynucleotide of the present invention can routinely be used as achromosome marker using techniques known in the art.

[0379] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably at least 15 bp (e.g., 15-25 bp) from the sequencesshown in SEQ ID NO:X. Primers can optionally be selected using computeranalysis so that primers do not span more than one predicted exon in thegenomic DNA. These primers are then used for PCR screening of somaticcell hybrids containing individual human chromosomes. Only those hybridscontaining the human gene corresponding to SEQ ID NO:X will yield anamplified fragment.

[0380] Similarly, somatic hybrids provide a rapid method of PCR mappingthe polynucleotides to particular chromosomes. Three or more clones canbe assigned per day using a single thermal cycler. Moreover,sublocalization of the polynucleotides can be achieved with panels ofspecific chromosome fragments. Other gene mapping strategies that can beused include in situ hybridization, prescreening with labeled flowsorted chromosomes, preselection by hybridization to constructchromosome specific-cDNA libraries, and computer mapping techniques(See, e.g., Shuler, Trends Biotechnol 16:456-459 (1998) which is herebyincorporated by reference in its entirety).

[0381] Precise chromosomal location of the polynucleotides can also beachieved using fluorescence in situ hybridization (FISH) of a metaphasechromosomal spread. This technique uses polynucleotides as short as 500or 600 bases; however, polynucleotides 2,000-4,000 bp are preferred. Fora review of this technique, see Verma et al., “Human Chromosomes: aManual of Basic Techniques,” Pergamon Press, New York (1988).

[0382] For chromosome mapping, the polynucleotides can be usedindividually (to mark a single chromosome or a single site on thatchromosome) or in panels (for marking multiple sites and/or multiplechromosomes).

[0383] Thus, the present invention also provides a method forchromosomal localization which involves (a) preparing PCR primers fromthe polynucleotide sequences in Table 1 and SEQ ID NO:X and (b)screening somatic cell hybrids containing individual chromosomes.

[0384] The polynucleotides of the present invention would likewise beuseful for radiation hybrid mapping, HAPPY mapping, and long rangerestriction mapping. For a review of these techniques and others knownin the art, see, e.g. Dear, “Genome Mapping: A Practical Approach,” IRLPress at Oxford University Press, London (1997); Aydin, J. Mol. Med.77:691-694 (1999); Hacia et al., Mol. Psychiatry 3:483-492 (1998);Herrick et al., Chromosome Res. 7:409-423 (1999); Hamilton et al.,Methods Cell Biol. 62:265-280 (2000); and/or Ott, J. Hered. 90:68-70(1999) each of which is hereby incorporated by reference in itsentirety.

[0385] Once a polynucleotide has been mapped to a precise chromosomallocation, the physical position of the polynucleotide can be used inlinkage analysis. Linkage analysis establishes coinheritance between achromosomal location and presentation of a particular disease. (Diseasemapping data are found, for example, in V. McKusick, MendelianInheritance in Man (available on line through Johns Hopkins UniversityWelch Medical Library).) Assuming 1 megabase mapping resolution and onegene per 20 kb, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of 50-500 potential causativegenes.

[0386] Thus, once coinheritance is established, differences in apolynucleotide of the invention and the corresponding gene betweenaffected and unaffected individuals can be examined. First, visiblestructural alterations in the chromosomes, such as deletions ortranslocations, are examined in chromosome spreads or by PCR. If nostructural alterations exist, the presence of point mutations areascertained. Mutations observed in some or all affected individuals, butnot in normal individuals, indicates that the mutation may cause thedisease. However, complete sequencing of the polypeptide and thecorresponding gene from several normal individuals is required todistinguish the mutation from a polymorphism. If a new polymorphism isidentified, this polymorphic polypeptide can be used for further linkageanalysis.

[0387] Furthermore, increased or decreased expression of the gene inaffected individuals as compared to unaffected individuals can beassessed using the polynucleotides of the invention. Any of thesealterations (altered expression, chromosomal rearrangement, or mutation)can be used as a diagnostic or prognostic marker.

[0388] Thus, the invention also provides a diagnostic method usefulduring diagnosis of a disorder, involving measuring the expression levelof polynucleotides of the present invention in cells or body fluid froman individual and comparing the measured gene expression level with astandard level of polynucleotide expression level, whereby an increaseor decrease in the gene expression level compared to the standard isindicative of a disorder.

[0389] In still another embodiment, the invention includes a kit foranalyzing samples for the presence of proliferative and/or cancerouspolynucleotides derived from a test subject. In a general embodiment,the kit includes at least one polynucleotide probe containing anucleotide sequence that will specifically hybridize with apolynucleotide of the invention and a suitable container. In a specificembodiment, the kit includes two polynucleotide probes defining aninternal region of the polynucleotide of the invention, where each probehas one strand containing a 31′mer-end internal to the region. In afurther embodiment, the probes may be useful as primers for polymerasechain reaction amplification.

[0390] Where a diagnosis of a related disorder, including, for example,diagnosis of a tumor, has already been made according to conventionalmethods, the present invention is useful as a prognostic indicator,whereby patients exhibiting enhanced or depressed polynucleotide of theinvention expression will experience a worse clinical outcome relativeto patients expressing the gene at a level nearer the standard level.

[0391] By “measuring the expression level of polynucleotides of theinvention” is intended qualitatively or quantitatively measuring orestimating the level of the polypeptide of the invention or the level ofthe mRNA encoding the polypeptide of the invention in a first biologicalsample either directly (e.g., by determining or estimating absoluteprotein level or mRNA level) or relatively (e.g., by comparing to thepolypeptide level or mRNA level in a second biological sample).Preferably, the polypeptide level or mRNA level in the first biologicalsample is measured or estimated and compared to a standard polypeptidelevel or mRNA level, the standard being taken from a second biologicalsample obtained from an individual not having the related disorder orbeing determined by averaging levels from a population of individualsnot having a related disorder. As will be appreciated in the art, once astandard polypeptide level or mRNA level is known, it can be usedrepeatedly as a standard for comparison.

[0392] By “biological sample” is intended any biological sample obtainedfrom an individual, body fluid, cell line, tissue culture, or othersource which contains polypeptide of the present invention or thecorresponding mRNA. As indicated, biological samples include body fluids(such as semen, lymph, sera, plasma, urine, synovial fluid and spinalfluid) which contain the polypeptide of the present invention, andtissue sources found to express the polypeptide of the presentinvention. Methods for obtaining tissue biopsies and body fluids frommammals are well known in the art. Where the biological sample is toinclude mRNA, a tissue biopsy is the preferred source.

[0393] The method(s) provided above may preferrably be applied in adiagnostic method and/or kits in which polynucleotides and/orpolypeptides of the invention are attached to a solid support. In oneexemplary method, the support may be a “gene chip” or a “biologicalchip” as described in U.S. Pat. Nos. 5,837,832, 5,874,219, and5,856,174. Further, such a gene chip with polynucleotides of theinvention attached may be used to identify polymorphisms between theisolated polynucleotide sequences of the invention, with polynucleotidesisolated from a test subject. The knowledge of such polymorphisms (i.e.their location, as well as, their existence) would be beneficial inidentifying disease loci for many disorders, such as for example, inneural disorders, inmmune system disorders, muscular disorders,reproductive disorders, gastrointestinal disorders, pulmonary disorders,cardiovascular disorders, renal disorders, proliferative disorders,and/or cancerous diseases and conditions. Such a method is described inU.S. Pat. Nos. 5,858,659 and 5,856,104. The US Patents referenced supraare hereby incorporated by reference in their entirety herein.

[0394] The present invention encompasses polynucleotides of the presentinvention that are chemically synthesized, or reproduced as peptidenucleic acids (PNA), or according to other methods known in the art. Theuse of PNAs would serve as the preferred form if the polynucleotides ofthe invention are incorporated onto a solid support, or gene chip. Forthe purposes of the present invention, a peptide nucleic acid (PNA) is apolyamide type of DNA analog and the monomeric units for adenine,guanine, thymine and cytosine are available commercially (PerceptiveBiosystems). Certain components of DNA, such as phosphorus, phosphorusoxides, or deoxyribose derivatives, are not present in PNAs. Asdisclosed by P. E. Nielsen, M. Eghohn, R. H. Berg and O. Buchardt,Science 254, 1497 (1991); and M. Egholm, O. Buchardt, L. Christensen, C.Behrens, S. M. Freier, D. A. Driver, R. H. Berg, S. K. Kim, B. Norden,and P. E. Nielsen, Nature 365, 666 (1993), PNAs bind specifically andtightly to complementary DNA strands and are not degraded by nucleases.In fact, PNA binds more strongly to DNA than DNA itself does. This isprobably because there is no electrostatic repulsion between the twostrands, and also the polyamide backbone is more flexible. Because ofthis, PNA/DNA duplexes bind under a wider range of stringency conditionsthan DNA/DNA duplexes, making it easier to perform multiplexhybridization. Smaller probes can be used than with DNA due to thestrong binding. In addition, it is more likely that single basemismatches can be determined with PNA/DNA hybridization because a singlemismatch in a PNA/DNA 15-mer lowers the melting point (T.sub.m) by8°-20° C., vs. 4°-16° C. for the DNA/DNA 15-mer duplex. Also, theabsence of charge groups in PNA means that hybridization can be done atlow ionic strengths and reduce possible interference by salt during theanalysis.

[0395] The present invention have uses which include, but are notlimited to, detecting cancer in mammals. In particular the invention isuseful during diagnosis of pathological cell proliferative neoplasiaswhich include, but are not limited to: acute myelogenous leukemiasincluding acute monocytic leukemia, acute myeloblastic leukemia, acutepromyelocytic leukemia, acute myelomonocytic leukemia, acuteerythroleukemia, acute megakaryocytic leukemia, and acuteundifferentiated leukemia, etc.; and chronic myelogenous leukemiasincluding chronic myelomonocytic leukemia, chronic granulocyticleukemia, etc. Preferred mammals include monkeys, apes, cats, dogs,cows, pigs, horses, rabbits and humans. Particularly preferred arehumans.

[0396] Pathological cell proliferative disorders are often associatedwith inappropriate activation of proto-oncogenes. (Gelnann, E. P. etal., “The Etiology of Acute Leukemia: Molecular Genetics and ViralOncology,” in Neoplastic Diseases of the Blood, Vol 1., Wiemik, P. H. etal. eds., 161-182 (1985)). Neoplasias are now believed to result fromthe qualitative alteration of a normal cellular gene product, or fromthe quantitative modification of gene expression by insertion into thechromosome of a viral sequence, by chromosomal translocation of a geneto a more actively transcribed region, or by some other mechanism.(Gelmann et al., supra) It is likely that mutated or altered expressionof specific genes is involved in the pathogenesis of some leukemias,among other tissues and cell types. (Gelmann et al., supra) Indeed, thehuman counterparts of the oncogenes involved in some animal neoplasiashave been amplified or translocated in some cases of human leukemia andcarcinoma. (Gelmann et al., supra)

[0397] For example, c-myc expression is highly amplified in thenon-lymphocytic leukemia cell line HL-60. When HL-60 cells arechemically induced to stop proliferation, the level of c-myc is found tobe downregulated. (International Publication Number WO 91/15580).However, it has been shown that exposure of HL-60 cells to a DNAconstruct that is complementary to the 5′ end of c-myc or c-myb blockstranslation of the corresponding mRNAs which downregulates expression ofthe c-myc or c-myb proteins and causes arrest of cell proliferation anddifferentiation of the treated cells. (International Publication NumberWO 91/15580; Wickstrom et al., Proc. Natl. Acad. Sci. 85:1028 (1988);Anfossi et al., Proc. Natl. Acad. Sci. 86:3379 (1989)). However, theskilled artisan would appreciate the present invention's usefulness isnot be limited to treatment of proliferative disorders of hematopoieticcells and tissues, in light of the numerous cells and cell types ofvarying origins which are known to exhibit proliferative phenotypes.

[0398] In addition to the foregoing, a polynucleotide of the presentinvention can be used to control gene expression through triple helixformation or through antisense DNA or RNA. Antisense techniques arediscussed, for example, in Okano, J. Neurochem. 56: 560 (1991);“Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988). Triple helix formation is discussed in,for instance Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooneyet al., Science 241: 456 (1988); and Dervan et al., Science 251: 1360(1991). Both methods rely on binding of the polynucleotide to acomplementary DNA or RNA. For these techniques, preferredpolynucleotides are usually oligonucleotides 20 to 40 bases in lengthand complementary to either the region of the gene involved intranscription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073(1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1360 (1991) ) or to the mRNA itself (antisense—Okano, J.Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988).) Triple helixformation optimally results in a shut-off of RNA transcription from DNA,while antisense RNA hybridization blocks translation of an mRNA moleculeinto polypeptide. The oligonucleotide described above can also bedelivered to cells such that the antisense RNA or DNA may be expressedin vivo to inhibit production of polypeptide of the present inventionantigens. Both techniques are effective in model systems, and theinformation disclosed herein can be used to design antisense or triplehelix polynucleotides in an effort to treat disease, and in particular,for the treatment of proliferative diseases and/or conditions.

[0399] Polynucleotides of the present invention are also useful in genetherapy. One goal of gene therapy is to insert a normal gene into anorganism having a defective gene, in an effort to correct the geneticdefect. The polynucleotides disclosed in the present invention offer ameans of targeting such genetic defects in a highly accurate manner.Another goal is to insert a new gene that was not present in the hostgenome, thereby producing a new trait in the host cell.

[0400] The polynucleotides are also useful for identifying individualsfrom minute biological samples. The United States military, for example,is considering the use of restriction fragment length polymorphism(RFLP) for identification of its personnel. In this technique, anindividual's genomic DNA is digested with one or more restrictionenzymes, and probed on a Southern blot to yield unique bands foridentifying personnel. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched, or stolen, makingpositive identification difficult. The polynucleotides of the presentinvention can be used as additional DNA markers for RFLP.

[0401] The polynucleotides of the present invention can also be used asan alternative to RFLP, by determining the actual base-by-base DNAsequence of selected portions of an individual's genome. These sequencescan be used to prepare PCR primers for amplifying and isolating suchselected DNA, which can then be sequenced. Using this technique,individuals can be identified because each individual will have a uniqueset of DNA sequences. Once an unique ID database is established for anindividual, positive identification of that individual, living or dead,can be made from extremely small tissue samples.

[0402] Forensic biology also benefits from using DNA-basedidentification techniques as disclosed herein. DNA sequences taken fromvery small biological samples such as tissues, e.g., hair or skin, orbody fluids, e.g., blood, saliva, semen, synovial fluid, amniotic fluid,breast milk, lymph, pulmonary sputum or surfactant, urine, fecal matter,etc., can be amplified using PCR. In one prior art technique, genesequences amplified from polymorphic loci, such as DQa class II HLAgene, are used in forensic biology to identify individuals. (Erlich, H.,PCR Technology, Freeman and Co. (1992).) Once these specific polymorphicloci are amplified, they are digested with one or more restrictionenzymes, yielding an identifying set of bands on a Southern blot probedwith DNA corresponding to the DQa class II HLA gene. Similarly,polynucleotides of the present invention can be used as polymorphicmarkers for forensic purposes.

[0403] There is also a need for reagents capable of identifying thesource of a particular tissue. Such need arises, for example, inforensics when presented with tissue of unknown origin. Appropriatereagents can comprise, for example, DNA probes or primers prepared fromthe sequences of the present invention. Panels of such reagents canidentify tissue by species and/or by organ type. In a similar fashion,these reagents can be used to screen tissue cultures for contamination.

[0404] The polynucleotides of the present invention are also useful ashybridization probes for differential identification of the tissue(s) orcell type(s) present in a biological sample. Similarly, polypeptides andantibodies directed to polypeptides of the present invention are usefulto provide immunological probes for differential identification of thetissue(s) (e.g., immunohistochemistry assays) or cell type(s) (e.g.,immunocytochemistry assays). In addition, for a number of disorders ofthe above tissues or cells, significantly higher or lower levels of geneexpression of the polynucleotides/polypeptides of the present inventionmay be detected in certain tissues (e.g., tissues expressingpolypeptides and/or polynucleotides of the present invention and/orcancerous and/or wounded tissues) or bodily fluids (e.g., serum, plasma,urine, synovial fluid or spinal fluid) taken from an individual havingsuch a disorder, relative to a “standard” gene expression level, i.e.,the expression level in healthy tissue from an individual not having thedisorder.

[0405] Thus, the invention provides a diagnostic method of a disorder,which involves: (a) assaying gene expression level in cells or bodyfluid of an individual; (b) comparing the gene expression level with astandard gene expression level, whereby an increase or decrease in theassayed gene expression level compared to the standard expression levelis indicative of a disorder.

[0406] In the very least, the polynucleotides of the present inventioncan be used as molecular weight markers on Southern gels, as diagnosticprobes for the presence of a specific mRNA in a particular cell type, asa probe to “subtract-out” known sequences in the process of discoveringnovel polynucleotides, for selecting and making oligomers for attachmentto a “gene chip” or other support, to raise anti-DNA antibodies usingDNA immunization techniques, and as an antigen to elicit an immuneresponse.

[0407] Uses of the Polypeptides

[0408] Each of the polypeptides identified herein can be used innumerous ways. The following description should be considered exemplaryand utilizes known techniques.

[0409] Polypeptides and antibodies directed to polypeptides of thepresent invention are useful to provide immunological probes fordifferential identification of the tissue(s) (e.g., immunohistochemistryassays such as, for example, ABC immunoperoxidase (Hsu et al., J.Histochem. Cytochem. 29:577-580 (1981)) or cell type(s) (e.g.,immunocytochemistry assays).

[0410] Antibodies can be used to assay levels of polypeptides encoded bypolynucleotides of the invention in a biological sample using classicalimmunohistological methods known to those of skill in the art (e.g., seeJalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al.,J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methodsuseful for detecting protein gene expression include immunoassays, suchas the enzyme linked immunosorbent assay (ELISA) and theradioimmunoassay (RIA). Suitable antibody assay labels are known in theart and include enzyme labels, such as, glucose oxidase; radioisotopes,such as iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S),tritium (³H), indium (^(115m)In, ^(113m)In, ¹¹²In, ¹¹¹In), andtechnetium (⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga),palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F),¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, 140La, 175Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷SC, ¹⁸⁶Re,¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru; luminescent labels, such as luminol; andfluorescent labels, such as fluorescein and rhodamine, and biotin.

[0411] In addition to assaying levels of polypeptide of the presentinvention in a biological sample, proteins can also be detected in vivoby imaging. Antibody labels or markers for in vivo imaging of proteininclude those detectable by X-radiography, NMR or ESR. ForX-radiography, suitable labels include radioisotopes such as barium orcesium, which emit detectable radiation but are not overtly harmful tothe subject. Suitable markers for NMR and ESR include those with adetectable characteristic spin, such as deuterium, which may beincorporated into the antibody by labeling of nutrients for the relevanthybridoma.

[0412] A protein-specific antibody or antibody fragment which has beenlabeled with an appropriate detectable imaging moiety, such as aradioisotope (for example, ¹³¹I, ¹¹²In, ^(99m)Tc, (¹³¹I, ¹²⁵I, ¹²³I,¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (^(115m)In,^(113m)In, ¹¹²In, ¹¹¹In), and technetium (⁹⁹Tc, ^(99m)Tc), thallium(²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo),xenon (¹³³Xe), fluorine (¹⁸F, ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁹La, ¹⁷⁵Yb,¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru), a radio-opaquesubstance, or a material detectable by nuclear magnetic resonance, isintroduced (for example, parenterally, subcutaneously orintraperitoneally) into the mammal to be examined for immune systemdisorder. It will be understood in the art that the size of the subjectand the imaging system used will determine the quantity of imagingmoiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of ^(99m)Tc.The labeled antibody or antibody fragment will then preferentiallyaccumulate at the location of cells which express the polypeptideencoded by a polynucleotide of the invention. In vivo tumor imaging isdescribed in S. W. Burchiel et al., “Immunopharmacokinetics ofRadiolabeled Antibodies and Their Fragments” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982)).

[0413] In one embodiment, the invention provides a method for thespecific delivery of compositions of the invention to cells byadministering polypeptides of the invention (e.g., polypeptides encodedby polynucleotides of the invention and/or antibodies) that areassociated with heterologous polypeptides or nucleic acids. In oneexample, the invention provides a method for delivering a therapeuticprotein into the targeted cell. In another example, the inventionprovides a method for delivering a single stranded nucleic acid (e.g.,antisense or ribozymes) or double stranded nucleic acid (e.g., DNA thatcan integrate into the cell's genome or replicate episomally and thatcan be transcribed) into the targeted cell.

[0414] In another embodiment, the invention provides a method for thespecific destruction of cells (e.g., the destruction of tumor cells) byadministering polypeptides of the invention in association with toxinsor cytotoxic prodrugs.

[0415] By “toxin” is meant one or more compounds that bind and activateendogenous cytotoxic effector systems, radioisotopes, holotoxins,modified toxins, catalytic subunits of toxins, or any molecules orenzymes not normally present in or on the surface of a cell that underdefined conditions cause the cell's death. Toxins that may be usedaccording to the methods of the invention include, but are not limitedto, radioisotopes known in the art, compounds such as, for example,antibodies (or complement fixing containing portions thereof) that bindan inherent or induced endogenous cytotoxic effector system, thymidinekinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonasexotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweedantiviral protein, alpha-sarcin and cholera toxin. “Toxin” also includesa cytostatic or cytocidal agent, a therapeutic agent or a radioactivemetal ion, e.g., alpha-emitters such as, for example, ²¹³Bi, or otherradioisotopes such as, for example, ¹⁰³Pd, ¹³³Xe, ¹³¹I, ⁶⁸Ge, ⁵⁷Co,⁶⁵Zn, ⁸⁵Sr, ³²P, ³⁵S, ⁹⁰Y, ¹⁵³Sm, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn,⁹⁰Yttrium, ¹¹⁷Tin, ¹⁵⁶Rhenium, ¹⁶⁶Holmium, and ¹⁸⁸Rhenium; luminescentlabels, such as luminol; and fluorescent labels, such as fluorescein andrhodamine, and biotin.

[0416] Techniques known in the art may be applied to label polypeptidesof the invention (including antibodies). Such techniques include, butare not limited to, the use of bifunctional conjugating agents (seee.g., U.S. Pat. Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361;5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119;4,994,560; and 5,808,003; the contents of each of which are herebyincorporated by reference in its entirety).

[0417] Thus, the invention provides a diagnostic method of a disorder,which involves (a) assaying the expression level of a polypeptide of thepresent invention in cells or body fluid of an individual; and (b)comparing the assayed polypeptide expression level with a standardpolypeptide expression level, whereby an increase or decrease in theassayed polypeptide expression level compared to the standard expressionlevel is indicative of a disorder. With respect to cancer, the presenceof a relatively high amount of transcript in biopsied tissue from anindividual may indicate a predisposition for the development of thedisease, or may provide a means for detecting the disease prior to theappearance of actual clinical symptoms. A more definitive diagnosis ofthis type may allow health professionals to employ preventative measuresor aggressive treatment earlier thereby preventing the development orfurther progression of the cancer.

[0418] Moreover, polypeptides of the present invention can be used totreat or prevent diseases or conditions such as, for example, neuraldisorders, immune system disorders, muscular disorders, reproductivedisorders, gastrointestinal disorders, pulmonary disorders,cardiovascular disorders, renal disorders, proliferative disorders,and/or cancerous diseases and conditions. For example, patients can beadministered a polypeptide of the present invention in an effort toreplace absent or decreased levels of the polypeptide (e.g., insulin),to supplement absent or decreased levels of a different polypeptide(e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repairproteins), to inhibit the activity of a polypeptide (e.g., an oncogeneor tumor supressor), to activate the activity of a polypeptide (e.g., bybinding to a receptor), to reduce the activity of a membrane boundreceptor by competing with it for free ligand (e.g., soluble TNFreceptors used in reducing inflammation), or to bring about a desiredresponse (e.g., blood vessel growth inhibition, enhancement of theimmune response to proliferative cells or tissues).

[0419] Similarly, antibodies directed to a polypeptide of the presentinvention can also be used to treat disease (as described supra, andelsewhere herein). For example, administration of an antibody directedto a polypeptide of the present invention can bind, and/or neutralizethe polypeptide, and/or reduce overproduction of the polypeptide.Similarly, administration of an antibody can activate the polypeptide,such as by binding to a polypeptide bound to a membrane (receptor).

[0420] At the very least, the polypeptides of the present invention canbe used as molecular weight markers on SDS-PAGE gels or on molecularsieve gel filtration columns using methods well known to those of skillin the art. Polypeptides can also be used to raise antibodies, which inturn are used to measure protein expression from a recombinant cell, asa way of assessing transformation of the host cell. Moreover, thepolypeptides of the present invention can be used to test the followingbiological activities.

[0421] Gene Therapy Methods

[0422] Another aspect of the present invention is to gene therapymethods for treating or preventing disorders, diseases and conditions.The gene therapy methods relate to the introduction of nucleic acid(DNA, RNA and antisense DNA or RNA) sequences into an animal to achieveexpression of the polypeptide of the present invention. This methodrequires a polynucleotide which codes for a polypeptide of the presentinvention operatively linked to a promoter and any other geneticelements necessary for the expression of the polypeptide by the targettissue. Such gene therapy and delivery techniques are known in the art,see, for example, WO90/11092, which is herein incorporated by reference.

[0423] Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) comprising a promoter operably linked to apolynucleotide of the present invention ex vivo, with the engineeredcells then being provided to a patient to be treated with thepolypeptide of the present invention. Such methods are well-known in theart. For example, see Belldegrun, A., et al., J. Natl. Cancer Inst. 85:207-216 (1993); Ferrantini, M. et al., Cancer Research 53: 1107-1112(1993); Ferrantini, M. et al., J. Immunology 153: 4604-4615 (1994);Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995); Ogura, H., et al.,Cancer Research 50: 5102-5106 (1990); Santodonato, L., et al., HumanGene Therapy 7:1-10 (1996); Santodonato, L., et al., Gene Therapy4:1246-1255 (1997); and Zhang, J.-F. et al., Cancer Gene Therapy 3:31-38 (1996)), which are herein incorporated by reference. In oneembodiment, the cells which are engineered are arterial cells. Thearterial cells may be reintroduced into the patient through directinjection to the artery, the tissues surrounding the artery, or throughcatheter injection.

[0424] As discussed in more detail below, the polynucleotide constructscan be delivered by any method that delivers injectable materials to thecells of an animal, such as, injection into the interstitial space oftissues (heart, muscle, skin, lung, liver, and the like). Thepolynucleotide constructs may be delivered in a pharmaceuticallyacceptable liquid or aqueous carrier.

[0425] In one embodiment, the polynucleotide of the present invention isdelivered as a naked polynucleotide. The term “naked” polynucleotide,DNA or RNA refers to sequences that are free from any delivery vehiclethat acts to assist, promote or facilitate entry into the cell,including viral sequences, viral particles, liposome formulations,lipofectin or precipitating agents and the like. However, thepolynucleotide of the present invention can also be delivered inliposome formulations and lipofectin formulations and the like can beprepared by methods well known to those skilled in the art. Such methodsare described, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and5,580,859, which are herein incorporated by reference.

[0426] The polynucleotide vector constructs used in the gene therapymethod are preferably constructs that will not integrate into the hostgenome nor will they contain sequences that allow for replication.Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSGavailable from Stratagene; pSVK3, pBPV, pMSG and pSVL available fromPharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available fromInvitrogen. Other suitable vectors will be readily apparent to theskilled artisan.

[0427] Any strong promoter known to those skilled in the art can be usedfor driving the expression of the polynucleotide sequence. Suitablepromoters include adenoviral promoters, such as the adenoviral majorlate promoter; or heterologous promoters, such as the cytomegalovirus(CMV) promoter; the respiratory syncytial virus (RSV) promoter;inducible promoters, such as the MMT promoter, the metallothioneinpromoter; heat shock promoters; the albumin promoter; the ApoAIpromoter; human globin promoters; viral thymidine kinase promoters, suchas the Herpes Simplex thymidine kinase promoter; retroviral LTRs; theb-actin promoter; and human growth hormone promoters. The promoter alsomay be the native promoter for the polynucleotide of the presentinvention.

[0428] Unlike other gene therapy techniques, one major advantage ofintroducing naked nucleic acid sequences into target cells is thetransitory nature of the polynucleotide synthesis in the cells. Studieshave shown that non-replicating DNA sequences can be introduced intocells to provide production of the desired polypeptide for periods of upto six months.

[0429] The polynucleotide construct can be delivered to the interstitialspace of tissues within the an animal, including of muscle, skin, brain,lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellular,fluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

[0430] For the naked nucleic acid sequence injection, an effectivedosage amount of DNA or RNA will be in the range of from about 0.05mg/kg body weight to about 50 mg/kg body weight. Preferably the dosagewill be from about 0.005 mg/kg to about 20 mg/kg and more preferablyfrom about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan ofordinary skill will appreciate, this dosage will vary according to thetissue site of injection. The appropriate and effective dosage ofnucleic acid sequence can readily be determined by those of ordinaryskill in the art and may depend on the condition being treated and theroute of administration.

[0431] The preferred route of administration is by the parenteral routeof injection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked DNAconstructs can be delivered to arteries during angioplasty by thecatheter used in the procedure.

[0432] The naked polynucleotides are delivered by any method known inthe art, including, but not limited to, direct needle injection at thedelivery site, intravenous injection, topical administration, catheterinfusion, and so-called “gene guns”. These delivery methods are known inthe art.

[0433] The constructs may also be delivered with delivery vehicles suchas viral sequences, viral particles, liposome formulations, lipofectin,precipitating agents, etc. Such methods of delivery are known in theart.

[0434] In certain embodiments, the polynucleotide constructs arecomplexed in a liposome preparation. Liposomal preparations for use inthe instant invention include cationic (positively charged), anionic(negatively charged) and neutral preparations. However, cationicliposomes are particularly preferred because a tight charge complex canbe formed between the cationic liposome and the polyanionic nucleicacid. Cationic liposomes have been shown to mediate intracellulardelivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA(1987) 84:7413-7416, which is herein incorporated by reference); mRNA(Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081, which isherein incorporated by reference); and purified transcription factors(Debs et al., J. Biol. Chem. (1990) 265:10189-10192, which is hereinincorporated by reference), in functional form.

[0435] Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areparticularly useful and are available under the trademark Lipofectin,from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc.Natl Acad. Sci. USA (1987) 84:7413-7416, which is herein incorporated byreference). Other commercially available liposomes include transfectace(DDAB/DOPE) and DOTAP/DOPE (Boehringer).

[0436] Other cationic liposomes can be prepared from readily availablematerials using techniques well known in the art. See, e.g. PCTPublication No. WO 90/11092 (which is herein incorporated by reference)for a description of the synthesis of DOTAP(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparationof DOTMA liposomes is explained in the literature, see, e.g., P. Felgneret al., Proc. Natl. Acad. Sci. USA 84:7413-7417, which is hereinincorporated by reference. Similar methods can be used to prepareliposomes from other cationic lipid materials.

[0437] Similarly, anionic and neutral liposomes are readily available,such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easilyprepared using readily available materials. Such materials includephosphatidyl, choline, cholesterol, phosphatidyl ethanolamine,dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol(DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. Thesematerials can also be mixed with the DOTMA and DOTAP starting materialsin appropriate ratios. Methods for making liposomes using thesematerials are well known in the art.

[0438] For example, commercially dioleoylphosphatidyl choline (DOPC),dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations to makeconventional liposomes, with or without the addition of cholesterol.Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mgeach of DOPG and DOPC under a stream of nitrogen gas into a sonicationvial. The sample is placed under a vacuum pump overnight and is hydratedthe following day with deionized water. The sample is then sonicated for2 hours in a capped vial, using a Heat Systems model 350 sonicatorequipped with an inverted cup (bath type) probe at the maximum settingwhile the bath is circulated at 15EC. Alternatively, negatively chargedvesicles can be prepared without sonication to produce multilamellarvesicles or by extrusion through nucleopore membranes to produceunilamellar vesicles of discrete size. Other methods are known andavailable to those of skill in the art.

[0439] The liposomes can comprise multilamellar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), withSUVs being preferred. The various liposome-nucleic acid complexes areprepared using methods well known in the art. See, e.g., Straubinger etal., Methods of Immunology (1983), 101:512-527, which is hereinincorporated by reference. For example, MLVs containing nucleic acid canbe prepared by depositing a thin film of phospholipid on the walls of aglass tube and subsequently hydrating with a solution of the material tobe encapsulated. SUVs are prepared by extended sonication of MLVs toproduce a homogeneous population of unilamellar liposomes. The materialto be entrapped is added to a suspension of preformed MLVs and thensonicated. When using liposomes containing cationic lipids, the driedlipid film is resuspended in an appropriate solution such as sterilewater or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated,and then the preformed liposomes are mixed directly with the DNA. Theliposome and DNA form a very stable complex due to binding of thepositively charged liposomes to the cationic DNA. SUVs find use withsmall nucleic acid fragments. LUVs are prepared by a number of methods,well known in the art. Commonly used methods include Ca²⁺-EDTA chelation(Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483; Wilsonet al., Cell (1979) 17:77); ether injection (Deamer, D. and Bangham, A.,Biochim. Biophys. Acta (1976) 443:629; Ostro et al., Biochem. Biophys.Res. Commun. (1977) 76:836; Fraley et al., Proc. Natl. Acad. Sci. USA(1979) 76:3348); detergent dialysis (Enoch, H. and Strittmatter, P.,Proc. Natl. Acad. Sci. USA (1979) 76:145); and reverse-phase evaporation(REV) (Fraley et al., J. Biol. Chem. (1980) 255:10431; Szoka, F. andPapahadjopoulos, D., Proc. Natl. Acad. Sci. USA (1978) 75:145;Schaefer-Ridder et al., Science (1982) 215:166), which are hereinincorporated by reference.

[0440] Generally, the ratio of DNA to liposomes will be from about 10:1to about 1:10. Preferably, the ration will be from about 5:1 to about1:5. More preferably, the ration will be about 3:1 to about 1:3. Stillmore preferably, the ratio will be about 1:1.

[0441] U.S. Pat. No. 5,676,954 (which is herein incorporated byreference) reports on the injection of genetic material, complexed withcationic liposomes carriers, into mice. U.S. Pat. Nos. 4,897,355,4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859,5,703,055, and international publication no. WO 94/9469 (which areherein incorporated by reference) provide cationic lipids for use intransfecting DNA into cells and mammals. U.S. Pat. Nos. 5,589,466,5,693,622, 5,580,859, 5,703,055, and international publication no. WO94/9469 (which are herein incorporated by reference) provide methods fordelivering DNA-cationic lipid complexes to mammals.

[0442] In certain embodiments, cells are engineered, ex vivo or in vivo,using a retroviral particle containing RNA which comprises a sequenceencoding a polypeptide of the present invention. Retroviruses from whichthe retroviral plasmid vectors may be derived include, but are notlimited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Roussarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon apeleukemia virus, human immunodeficiency virus, Myeloproliferative SarcomaVirus, and mammary tumor virus.

[0443] The retroviral plasmid vector is employed to transduce packagingcell lines to form producer cell lines. Examples of packaging cellswhich may be transfected include, but are not limited to, the PE501,PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86,GP+envAm12, and DAN cell lines as described in Miller, Human GeneTherapy 1:5-14 (1990), which is incorporated herein by reference in itsentirety. The vector may transduce the packaging cells through any meansknown in the art. Such means include, but are not limited to,electroporation, the use of liposomes, and CaPO₄ precipitation. In onealternative, the retroviral plasmid vector may be encapsulated into aliposome, or coupled to a lipid, and then administered to a host.

[0444] The producer cell line generates infectious retroviral vectorparticles which include polynucleotide encoding a polypeptide of thepresent invention. Such retroviral vector particles then may beemployed, to transduce eukaryotic cells, either in vitro or in vivo. Thetransduced eukaryotic cells will express a polypeptide of the presentinvention.

[0445] In certain other embodiments, cells are engineered, ex vivo or invivo, with polynucleotide contained in an adenovirus vector. Adenoviruscan be manipulated such that it encodes and expresses a polypeptide ofthe present invention, and at the same time is inactivated in terms ofits ability to replicate in a normal lytic viral life cycle. Adenovirusexpression is achieved without integration of the viral DNA into thehost cell chromosome, thereby alleviating concerns about insertionalmutagenesis. Furthermore, adenoviruses have been used as live entericvaccines for many years with an excellent safety profile (Schwartz, A.R. et al. (1974) Am. Rev. Respir. Dis.109:233-238). Finally, adenovirusmediated gene transfer has been demonstrated in a number of instancesincluding transfer of alpha-1-antitrypsin and CFTR to the lungs ofcotton rats (Rosenfeld, M. A. et al. (1991) Science 252:431-434;Rosenfeld et al., (1992) Cell 68:143-155). Furthermore, extensivestudies to attempt to establish adenovirus as a causative agent in humancancer were uniformly negative (Green, M. et al. (1979) Proc. Natl.Acad. Sci. USA 76:6606).

[0446] Suitable adenoviral vectors useful in the present invention aredescribed, for example, in Kozarsky and Wilson, Curr. Opin. Genet.Devel. 3:499-503 (1993); Rosenfeld et al., Cell 68:143-155 (1992);Engelhardt et al., Human Genet. Ther. 4:759-769 (1993); Yang et al.,Nature Genet. 7:362-369 (1994); Wilson et al., Nature 365:691-692(1993); and U.S. Pat. No. 5,652,224, which are herein incorporated byreference. For example, the adenovirus vector Ad2 is useful and can begrown in human 293 cells. These cells contain the E1 region ofadenovirus and constitutively express E1a and E1b, which complement thedefective adenoviruses by providing the products of the genes deletedfrom the vector. In addition to Ad2, other varieties of adenovirus(e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.

[0447] Preferably, the adenoviruses used in the present invention arereplication deficient. Replication deficient adenoviruses require theaid of a helper virus and/or packaging cell line to form infectiousparticles. The resulting virus is capable of infecting cells and canexpress a polynucleotide of interest which is operably linked to apromoter, but cannot replicate in most cells. Replication deficientadenoviruses may be deleted in one or more of all or a portion of thefollowing genes: E1a, E1b, E3, E4, E2a, or L1 through L5.

[0448] In certain other embodiments, the cells are engineered, ex vivoor in vivo, using an adeno-associated virus (AAV). AAVs are naturallyoccurring defective viruses that require helper viruses to produceinfectious particles (Muzyczka, N., Curr. Topics in Microbiol. Immunol.158:97 (1992)). It is also one of the few viruses that may integrate itsDNA into non-dividing cells. Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate, but space for exogenousDNA is limited to about 4.5 kb. Methods for producing and using suchAAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941,5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

[0449] For example, an appropriate AAV vector for use in the presentinvention will include all the sequences necessary for DNA replication,encapsidation, and host-cell integration. The polynucleotide constructis inserted into the AAV vector using standard cloning methods, such asthose found in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Press (1989). The recombinant AAV vector is thentransfected into packaging cells which are infected with a helper virus,using any standard technique, including lipofection, electroporation,calcium phosphate precipitation, etc. Appropriate helper viruses includeadenoviruses, cytomegaloviruses, vaccinia viruses, or herpes viruses.Once the packaging cells are transfected and infected, they will produceinfectious AAV viral particles which contain the polynucleotideconstruct. These viral particles are then used to transduce eukaryoticcells, either ex vivo or in vivo. The transduced cells will contain thepolynucleotide construct integrated into its genome, and will express apolypeptide of the invention.

[0450] Another method of gene therapy involves operably associatingheterologous control regions and endogenous polynucleotide sequences(e.g. encoding a polypeptide of the present invention) via homologousrecombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997;International Publication No. WO 96/29411, published Sep. 26, 1996;International Publication No. WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); andZijlstra et al., Nature 342:435-438 (1989). This method involves theactivation of a gene which is present in the target cells, but which isnot normally expressed in the cells, or is expressed at a lower levelthan desired.

[0451] Polynucleotide constructs are made, using standard techniquesknown in the art, which contain the promoter with targeting sequencesflanking the promoter. Suitable promoters are described herein. Thetargeting sequence is sufficiently complementary to an endogenoussequence to permit homologous recombination of the promoter-targetingsequence with the endogenous sequence. The targeting sequence will besufficiently near the 5′ end of the desired endogenous polynucleotidesequence so the promoter will be operably linked to the endogenoussequence upon homologous recombination.

[0452] The promoter and the targeting sequences can be amplified usingPCR. Preferably, the amplified promoter contains distinct restrictionenzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the firsttargeting sequence contains the same restriction enzyme site as the 5′end of the amplified promoter and the 5′ end of the second targetingsequence contains the same restriction site as the 3′ end of theamplified promoter. The amplified promoter and targeting sequences aredigested and ligated together.

[0453] The promoter-targeting sequence construct is delivered to thecells, either as naked polynucleotide, or in conjunction withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, whole viruses, lipofection, precipitating agents, etc.,described in more detail above. The P promoter-targeting sequence can bedelivered by any method, included direct needle injection, intravenousinjection, topical administration, catheter infusion, particleaccelerators, etc. The methods are described in more detail below.

[0454] The promoter-targeting sequence construct is taken up by cells.Homologous recombination between the construct and the endogenoussequence takes place, such that an endogenous sequence is placed underthe control of the promoter. The promoter then drives the expression ofthe endogenous sequence.

[0455] Preferably, the polynucleotide encoding a polypeptide of thepresent invention contains a secretory signal sequence that facilitatessecretion of the protein. Typically, the signal sequence is positionedin the coding region of the polynucleotide to be expressed towards or atthe 5′ end of the coding region. The signal sequence may be homologousor heterologous to the polynucleotide of interest and may be homologousor heterologous to the cells to be transfected. Additionally, the signalsequence may be chemically synthesized using methods known in the art.

[0456] Any mode of administration of any of the above-describedpolynucleotides constructs can be used so long as the mode results inthe expression of one or more molecules in an amount sufficient toprovide a therapeutic effect. This includes direct needle injection,systemic injection, catheter infusion, biolistic injectors, particleaccelerators (i.e., “gene guns”), gelfoam sponge depots, othercommercially available depot materials, osmotic pumps (e.g., Alzaminipumps), oral or suppositorial solid (tablet or pill) pharmaceuticalformulations, and decanting or topical applications during surgery. Forexample, direct injection of naked calcium phosphate-precipitatedplasmid into rat liver and rat spleen or a protein-coated plasmid intothe portal vein has resulted in gene expression of the foreign gene inthe rat livers (Kaneda et al., Science 243:375 (1989)).

[0457] A preferred method of local administration is by directinjection. Preferably, a recombinant molecule of the present inventioncomplexed with a delivery vehicle is administered by direct injectioninto or locally within the area of arteries. Administration of acomposition locally within the area of arteries refers to injecting thecomposition centimeters and preferably, millimeters within arteries.

[0458] Another method of local administration is to contact apolynucleotide construct of the present invention in or around asurgical wound. For example, a patient can undergo surgery and thepolynucleotide construct can be coated on the surface of tissue insidethe wound or the construct can be injected into areas of tissue insidethe wound.

[0459] Therapeutic compositions useful in systemic administration,include recombinant molecules of the present invention complexed to atargeted delivery vehicle of the present invention. Suitable deliveryvehicles for use with systemic administration comprise liposomescomprising ligands for targeting the vehicle to a particular site.

[0460] Preferred methods of systemic administration, include intravenousinjection, aerosol, oral and percutaneous (topical) delivery.Intravenous injections can be performed using methods standard in theart. Aerosol delivery can also be performed using methods standard inthe art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA189:11277-11281, 1992, which is incorporated herein by reference). Oraldelivery can be performed by complexing a polynucleotide construct ofthe present invention to a carrier capable of withstanding degradationby digestive enzymes in the gut of an animal. Examples of such carriers,include plastic capsules or tablets, such as those known in the art.Topical delivery can be performed by mixing a polynucleotide constructof the present invention with a lipophilic reagent (e.g., DMSO) that iscapable of passing into the skin.

[0461] Determining an effective amount of substance to be delivered candepend upon a number of factors including, for example, the chemicalstructure and biological activity of the substance, the age and weightof the animal, the precise condition requiring treatment and itsseverity, and the route of administration. The frequency of treatmentsdepends upon a number of factors, such as the amount of polynucleotideconstructs administered per dose, as well as the health and history ofthe subject. The precise amount, number of doses, and timing of doseswill be determined by the attending physician or veterinarian.

[0462] Therapeutic compositions of the present invention can beadministered to any animal, preferably to mammals and birds. Preferredmammals include humans, dogs, cats, mice, rats, rabbits sheep, cattle,horses and pigs, with humans being particularly preferred.

[0463] Biological Activities

[0464] Polynucleotides or polypeptides, or agonists or antagonists ofthe present invention, can be used in assays to test for one or morebiological activities. If these polynucleotides or polypeptides, oragonists or antagonists of the present invention, do exhibit activity ina particular assay, it is likely that these molecules may be involved inthe diseases associated with the biological activity. Thus, thepolynucleotides and polypeptides, and agonists or antagonists could beused to treat the associated disease.

[0465] Apoptosis related proteins are believed to be involved inbiological activities associated with activation, signaling, andfacilitation of apoptosis. Accordingly, compositions of the invention(including polynucleotides, polypeptides and antibodies of theinvention, and fragments and variants thereof) may be used in thediagnosis, detection and/or treatment of diseases and/or disordersassociated with aberrant apoptosis activity. In preferred embodiments,compositions of the invention (including polynucleotides, polypeptidesand antibodies of the invention, and fragments and variants thereof) maybe used in the diagnosis, detection and/or treatment of diseases and/ordisorders relating to apoptosis (e.g., apoptosis, and/or as describedunder “Immnune activity” and “Hyperproliferative Disorders” below),neoplasias of the Immune system (e.g., Lymphomas, leukemias, and/or asdescribed under “Immune Activity” and “Diseases at the Cellular Level”below), autoimmune disorders (e.g., rheumatoid arthritis, inflammatorydisorders, and/or as described under “Immune Activity” below), andneural disorders (e.g., as described under “Neural Activity andNeurological Diseases” below). Thus, polynucleotides, translationproducts and antibodies of the invention are useful in the diagnosis,detection and/or treatment of diseases and/or disorders associated withactivities that include, but are not limited to, apoptosis and apoptoticdisorders, neoplasias, inflammation, autoimmune disorders, and neuraldisorders.

[0466] More generally, polynucleotides, translation products andantibodies corresponding to this gene may be useful for the diagnosis,detection and/or treatment of diseases and/or disorders associated withthe following systems.

[0467] Immune Activity

[0468] Polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention may be useful in treating,preventing, and/or diagnosing diseases, disorders, and/or conditions ofthe immune system, by, for example, activating or inhibiting theproliferation, differentiation, or mobilization (chemotaxis) of immunecells. Immune cells develop through a process called hematopoiesis,producing myeloid (platelets, red blood cells, neutrophils, andmacrophages) and lymphoid (B and T lymphocytes) cells from pluripotentstem cells. The etiology of these immune diseases, disorders, and/orconditions may be genetic, somatic, such as cancer and some autoimmunediseases, acquired (e.g., by chemotherapy or toxins), or infectious.Moreover, polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention can be used as a marker or detectorof a particular immune system disease or disorder.

[0469] Polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention may be useful in treating,preventing, and/or diagnosing diseases, disorders, and/or conditions ofhematopoietic cells. Polynucleotides, polypeptides, antibodies, and/oragonists or antagonists of the present invention could be used toincrease differentiation and proliferation of hematopoietic cells,including the pluripotent stem cells, in an effort to treat or preventthose diseases, disorders, and/or conditions associated with a decreasein certain (or many) types hematopoietic cells. Examples of immunologicdeficiency syndromes include, but are not limited to: blood proteindiseases, disorders, and/or conditions (e.g., agammaglobulinemia,dysgammaglobulinemia), ataxia telangiectasia, common variableimmunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLV infection,leukocyte adhesion deficiency syndrome, lymphopenia, phagocytebactericidal dysfunction, severe combined immunodeficiency (SCIDs),Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or hemoglobinuria.

[0470] Moreover, polynucleotides, polypeptides, antibodies, and/oragonists or antagonists of the present invention could also be used tomodulate hemostatic (the stopping of bleeding) or thrombolytic activity(clot formation). For example, by increasing hemostatic or thrombolyticactivity, polynucleotides or polypeptides, and/or agonists orantagonists of the present invention could be used to treat or preventblood coagulation diseases, disorders, and/or conditions (e.g.,afibrinogenemia, factor deficiencies), blood platelet diseases,disorders, and/or conditions (e.g., thrombocytopenia), or woundsresulting from trauma, surgery, or other causes. Alternatively,polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention that can decrease hemostatic orthrombolytic activity could be used to inhibit or dissolve clotting.These molecules could be important in the treatment or prevention ofheart attacks (infarction), strokes, or scarring.

[0471] The polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention may be useful in treating,preventing, and/or diagnosing autoimmune disorders. Many autoimmunedisorders result from inappropriate recognition of self as foreignmaterial by immune cells. This inappropriate recognition results in animmune response leading to the destruction of the host tissue.Therefore, the administration of polynucleotides and polypeptides of theinvention that can inhibit an immune response, particularly theproliferation, differentiation, or chemotaxis of T-cells, may be aneffective therapy in preventing autoimmune disorders.

[0472] Autoimmune diseases or disorders that may be treated, prevented,and/or diagnosed by polynucleotides, polypeptides, antibodies, and/oragonists or antagonists of the present invention include, but are notlimited to, one or more of the following: autoimmune hemolytic anemia,autoimmune neonatal thrombocytopenia, idiopathic thrombocytopeniapurpura, autoimmunocytopenia, hemolytic anemia, antiphospholipidsyndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsingpolychondritis, rheumatic heart disease, glomerulonephritis (e.g, IgAnephropathy), Multiple Sclerosis, Neuritis, Uveitis Ophthalmia,Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura),Reiter's Disease, Stiff-Man Syndrome, Autoimmune Pulmonary Inflammation,Autism, Guillain-Barre Syndrome, insulin dependent diabetes mellitis,and autoimmune inflammatory eye, autoimmune thyroiditis, hypothyroidism(i.e., Hashimoto's thyroiditis, systemic lupus erhythematosus,Goodpasture's syndrome, Pemphigus, Receptor autoimmunities such as, forexample, (a) Graves' Disease, (b) Myasthenia Gravis, and (c) insulinresistance, autoimmune hemolytic anemia, autoimmune thrombocytopenicpurpura, rheumatoid arthritis, schleroderma with anti-collagenantibodies, mixed connective tissue disease,polymyositis/dermatomyositis, pernicious anemia, idiopathic Addison'sdisease, infertility, glomerulonephritis such as primaryglomerulonephritis and IgA nephropathy, bullous pemphigoid, Sjogren'ssyndrome, diabetes millitus, and adrenergic drug resistance (includingadrenergic drug resistance with asthma or cystic fibrosis), chronicactive hepatitis, primary biliary cirrhosis, other endocrine glandfailure, vitiligo, vasculitis, post-MI, cardiotomy syndrome, urticaria,atopic dermatitis, asthma, inflammatory myopathies, and otherinflammatory, granulamatous, degenerative, and atrophic disorders.

[0473] Additional autoimmune disorders (that are probable) that may betreated, prevented, and/or diagnosed with the compositions of theinvention include, but are not limited to, rheumatoid arthritis (oftencharacterized, e.g., by immune complexes in joints), scleroderma withanti-collagen antibodies (often characterized, e.g., by nucleolar andother nuclear antibodies), mixed connective tissue disease (oftencharacterized, e.g., by antibodies to extractable nuclear antigens(e.g., ribonucleoprotein)), polymyositis (often characterized, e.g., bynonhistone ANA), pernicious anemia (often characterized, e.g., byantiparietal cell, microsomes, and intrinsic factor antibodies),idiopathic Addison's disease (often characterized, e.g., by humoral andcell-mediated adrenal cytotoxicity, infertility (often characterized,e.g., by antispermatozoal antibodies), glomerulonephritis (oftencharacterized, e.g., by glomerular basement membrane antibodies orimmune complexes), bullous pemphigoid (often characterized, e.g., by IgGand complement in basement membrane), Sjogren's syndrome (oftencharacterized, e.g., by multiple tissue antibodies, and/or a specificnonhistone ANA (SS-B)), diabetes millitus (often characterized, e.g., bycell-mediated and humoral islet cell antibodies), and adrenergic drugresistance (including adrenergic drug resistance with asthma or cysticfibrosis) (often characterized, e.g., by beta-adrenergic receptorantibodies).

[0474] Additional autoimmune disorders (that are possible) that may betreated, prevented, and/or diagnosed with the compositions of theinvention include, but are not limited to, chronic active hepatitis(often characterized, e.g., by smooth muscle antibodies), primarybiliary cirrhosis (often characterized, e.g., by mitchondrialantibodies), other endocrine gland failure (often characterized, e.g.,by specific tissue antibodies in some cases), vitiligo (oftencharacterized, e.g., by melanocyte antibodies), vasculitis (oftencharacterized, e.g., by Ig and complement in vessel walls and/or lowserum complement), post-MI (often characterized, e.g., by myocardialantibodies), cardiotomy syndrome (often characterized, e.g., bymyocardial antibodies), urticaria (often characterized, e.g., by IgG andIgM antibodies to IgE), atopic dermatitis (often characterized, e.g., byIgG and IgM antibodies to IgE), asthma (often characterized, e.g., byIgG and IgM antibodies to IgE), and many other inflammatory,granulamatous, degenerative, and atrophic disorders.

[0475] In a preferred embodiment, the autoimmune diseases and disordersand/or conditions associated with the diseases and disorders recitedabove are treated, prevented, and/or diagnosed using for example,antagonists or agonists, polypeptides or polynucleotides, or antibodiesof the present invention.

[0476] In a preferred embodiment polynucleotides, polypeptides,antibodies, and/or agonists or antagonists of the present inventioncould be used as an agent to boost immunoresponsiveness among B celland/or T cell immunodeficient individuals.

[0477] B cell immunodeficiencies that may be ameliorated or treated byadministering the polypeptides or polynucleotides of the invention,and/or agonists thereof, include, but are not limited to, severecombined immunodeficiency (SCID)-X linked, SCID-autosomal, adenosinedeaminase deficiency (ADA deficiency), X-linked agammaglobulinemia(XLA), Bruton's disease, congenital agammaglobulinemia, X-linkedinfantile agammaglobulinemia, acquired agammaglobulinemia, adult onsetagammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia,hypogammaglobulinemia, transient hypogammaglobulinemia of infancy,unspecified hypogammaglobulinemia, agammaglobulinemia, common variableimmunodeficiency (CVI) (acquired), Wiskott-Aldrich Syndrome (WAS),X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiencywith hyper IgM, selective IgA deficiency, IgG subclass deficiency (withor without IgA deficiency), antibody deficiency with normal or elevatedIgs, immunodeficiency with thymoma, Ig heavy chain deletions, kappachain deficiency, B cell lymphoproliferative disorder (BLPD), selectiveIgM immunodeficiency, recessive agammaglobulinemia (Swiss type),reticular dysgenesis, neonatal neutropenia, severe congenitalleukopenia, thymic alymophoplasia-aplasia or dysplasia withimmunodeficiency, ataxia-telangiectasia, short limbed dwarfism, X-linkedlymphoproliferative syndrome (XLP), Nezelof syndrome-combinedimmunodeficiency with Igs, purine nucleoside phosphorylase deficiency(PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severecombined immunodeficiency.

[0478] T cell deficiencies that may be ameliorated or treated byadministering the polypeptides or polynucleotides of the invention,and/or agonists thereof include, but are not limited to, for example,DiGeorge anomaly, thymic hypoplasia, third and fourth pharyngeal pouchsyndrome, 22q11.2 deletion, chronic mucocutaneous candidiasis, naturalkiller cell deficiency (NK), idiopathic CD4+ T-lymphocytopenia,immunodeficiency with predominant T cell defect (unspecified), andunspecified immunodeficiency of cell mediated immunity. In specificembodiments, DiGeorge anomaly or conditions associated with DiGeorgeanomaly are ameliorated or treated by, for example, administering thepolypeptides or polynucleotides of the invention, or antagonists oragonists thereof.

[0479] Other immunodeficiencies that may be ameliorated or treated byadministering polypeptides or polynucleotides of the invention, and/oragonists thereof, include, but are not limited to, severe combinedimmunodeficiency (SCID; e.g., X-linked SCID, autosomal SCID, andadenosine deaminase deficiency), ataxia-telangiectasia, Wiskott-Aldrichsyndrome, short-limber dwarfism, X-linked lymphoproliferative syndrome(XLP), Nezelof syndrome (e.g., purine nucleoside phosphorylasedeficiency), MHC Class II deficiency. In specific embodiments,ataxia-telangiectasia or conditions associated withataxia-telangiectasia are ameliorated or treated by administering thepolypeptides or polynucleotides of the invention, and/or agoniststhereof.

[0480] In a specific preferred embodiment, rheumatoid arthritis istreated, prevented, and/or diagnosed using polynucleotides,polypeptides, antibodies, and/or agonists or antagonists of the presentinvention. In another specific preferred embodiment, systemic lupuserythemosus is treated, prevented, and/or diagnosed usingpolynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention. In another specific preferredembodiment, idiopathic thrombocytopenia purpura is treated, prevented,and/or diagnosed using polynucleotides, polypeptides, antibodies, and/oragonists or antagonists of the present invention. In another specificpreferred embodiment IgA nephropathy is treated, prevented, and/ordiagnosed using polynucleotides, polypeptides, antibodies, and/oragonists or antagonists of the present invention. In a preferredembodiment, the autoimmune diseases and disorders and/or conditionsassociated with the diseases and disorders recited above are treated,prevented, and/or diagnosed using antibodies against the protein of theinvention.

[0481] Similarly, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated, prevented, and/or diagnosed using polypeptides, antibodies,or polynucleotides of the invention, and/or agonists or antagoniststhereof. Moreover, these molecules can be used to treat, prevent, and/ordiagnose anaphylaxis, hypersensitivity to an antigenic molecule, orblood group incompatibility.

[0482] Moreover, inflammatory conditions may also be treated, diagnosed,and/or prevented with polynucleotides, polypeptides, antibodies, and/oragonists or antagonists of the present invention. Such inflammatoryconditions include, but are not limited to, for example, respiratorydisorders (such as, e.g., asthma and allergy); gastrointestinaldisorders (such as, e.g., inflammatory bowel disease); cancers (such as,e.g., gastric, ovarian, lung, bladder, liver, and breast); CNS disorders(such as, e.g., multiple sclerosis, blood-brain barrier permeability,ischemic brain injury and/or stroke, traumatic brain injury,neurodegenerative disorders (such as, e.g., Parkinson's disease andAlzheimer's disease), AIDS-related dementia, and prion disease);cardiovascular disorders (such as, e.g., atherosclerosis, myocarditis,cardiovascular disease, and cardiopulmonary bypass complications); aswell as many additional diseases, conditions, and disorders that arecharacterized by inflammation (such as, e.g., chronic hepatitis (B andC), rheumatoid arthritis, gout, trauma, septic shock, pancreatitis,sarcoidosis, dermatitis, renal ischemia-reperfusion injury, Grave'sdisease, systemic lupus erythematosis, diabetes mellitus (i.e., type 1diabetes), and allogenic transplant rejection).

[0483] In specific embodiments, polypeptides, antibodies, orpolynucleotides of the invention, and/or agonists or antagoniststhereof, are useful to treat, diagnose, and/or prevent transplantationrejections, graft-versus-host disease, autoimmune and inflammatorydiseases (e.g., immune complex-induced vasculitis, glomerulonephritis,hemolytic anemia, myasthenia gravis, type II collagen-induced arthritis,experimental allergic and hyperacute xenograft rejection, rheumatoidarthritis, and systemic lupus erythematosus (SLE). Organ rejectionoccurs by host immune cell destruction of the transplanted tissuethrough an immune response. Similarly, an immune response is alsoinvolved in GVHD, but, in this case, the foreign transplanted immunecells destroy the host tissues. Polypeptides, antibodies, orpolynucleotides of the invention, and/or agonists or antagoniststhereof, that inhibit an immune response, particularly the activation,proliferation, differentiation, or chemotaxis of T-cells, may be aneffective therapy in preventing organ rejection or GVHD.

[0484] Similarly, polynucleotides, polypeptides, antibodies, and/oragonists or antagonists of the present invention may also be used tomodulate and/or diagnose inflammation. For example, since polypeptides,antibodies, or polynucleotides of the invention, and/or agonists orantagonists of the invention may inhibit the activation, proliferationand/or differentiation of cells involved in an inflammatory response,these molecules can be used to treat, diagnose, or prognose,inflammatory conditions, both chronic and acute conditions, including,but not limited to, inflammation associated with infection (e.g., septicshock, sepsis, or systemic inflammatory response syndrome (SIRS)),ischemia-reperfusion injury, endotoxin lethality, arthritis,complement-mediated hyperacute rejection, nephritis, cytokine orchemokine induced lung injury, inflammatory bowel disease, Crohn'sdisease, and resulting from over production of cytokines (e.g., TNF orIL-1.).

[0485] Polypeptides, antibodies, polynucleotides and/or agonists orantagonists of the invention can be used to treat, detect, and/orprevent infectious agents. For example, by increasing the immuneresponse, particularly increasing the proliferation activation and/ordifferentiation of B and/or T cells, infectious diseases may be treated,detected, and/or prevented. The immune response may be increased byeither enhancing an existing immune response, or by initiating a newimmune response. Alternatively, polynucleotides, polypeptides,antibodies, and/or agonists or antagonists of the present invention mayalso directly inhibit the infectious agent (refer to section ofapplication listing infectious agents, etc), without necessarilyeliciting an immune response.

[0486] Additional preferred embodiments of the invention include, butare not limited to, the use of polypeptides, antibodies, polynucleotidesand/or agonists or antagonists in the following applications:

[0487] Administration to an animal (e.g., mouse, rat, rabbit, hamster,guinea pig, pigs, micro-pig, chicken, camel, goat, horse, cow, sheep,dog, cat, non-human primate, and human, most preferably human) to boostthe immune system to produce increased quantities of one or moreantibodies (e.g., IgG, IgA, IgM, and IgE), to induce higher affinityantibody production (e.g., IgG, IgA, IgM, and IgE), and/or to increasean immune response.

[0488] Administration to an animal (including, but not limited to, thoselisted above, and also including transgenic animals) incapable ofproducing functional endogenous antibody molecules or having anotherwise compromised endogenous immune system, but which is capable ofproducing human immunoglobulin molecules by means of a reconstituted orpartially reconstituted immune system from another animal (see, e.g.,published PCT Application Nos. WO98/24893, WO/9634096, WO/9633735, andWO/9110741.

[0489] A vaccine adjuvant that enhances immune responsiveness tospecific antigen.

[0490] An adjuvant to enhance tumor-specific immune responses.

[0491] An adjuvant to enhance anti-viral immune responses. Anti-viralimmune responses that may be enhanced using the compositions of theinvention as an adjuvant, include virus and virus associated diseases orsymptoms described herein or otherwise known in the art. In specificembodiments, the compositions of the invention are used as an adjuvantto enhance an immune response to a virus, disease, or symptom selectedfrom the group consisting of: AIDS, meningitis, Dengue, EBV, andhepatitis (e.g., hepatitis B). In another specific embodiment, thecompositions of the invention are used as an adjuvant to enhance animmune response to a virus, disease, or symptom selected from the groupconsisting of: HIV/AIDS, Respiratory syncytial virus, Dengue, Rotavirus,Japanese B encephalitis, Influenza A and B, Parainfluenza, Measles,Cytomegalovirus, Rabies, Junin, Chikungunya, Rift Valley fever, Herpessimplex, and yellow fever.

[0492] An adjuvant to enhance anti-bacterial or anti-fungal immuneresponses. Anti-bacterial or anti-fungal immune responses that may beenhanced using the compositions of the invention as an adjuvant, includebacteria or fungus and bacteria or fungus associated diseases orsymptoms described herein or otherwise known in the art. In specificembodiments, the compositions of the invention are used as an adjuvantto enhance an immune response to a bacteria or fungus, disease, orsymptom selected from the group consisting of: tetanus, Diphtheria,botulism, and meningitis type B. In another specific embodiment, thecompositions of the invention are used as an adjuvant to enhance animmune response to a bacteria or fungus, disease, or symptom selectedfrom the group consisting of: Vibrio cholerae, Mycobacterium leprae,Salmonella typhi, Salmonella paratyphi, Meisseria meningitidis,Streptococcus pneumoniae, Group B streptococcus, Shigella spp.,Enterotoxigenic Escherichia coli, Enterohemorrhagic E. coli, Borreliaburgdorferi, and Plasmodium (malaria).

[0493] An adjuvant to enhance anti-parasitic immune responses.Anti-parasitic immune responses that may be enhanced using thecompositions of the invention as an adjuvant, include parasite andparasite associated diseases or symptoms described herein or otherwiseknown in the art. In specific embodiments, the compositions of theinvention are used as an adjuvant to enhance an immune response to aparasite. In another specific embodiment, the compositions of theinvention are used as an adjuvant to enhance an immune response toPlasmodium (malaria).

[0494] As a stimulator of B cell responsiveness to pathogens.

[0495] As an activator of T cells.

[0496] As an agent that elevates the immune status of an individualprior to their receipt of immunosuppressive therapies.

[0497] As an agent to induce higher affinity antibodies.

[0498] As an agent to increase serum immunoglobulin concentrations.

[0499] As an agent to accelerate recovery of immunocompromisedindividuals.

[0500] As an agent to boost immunoresponsiveness among aged populations.

[0501] As an immune system enhancer prior to, during, or after bonemarrow transplant and/or other transplants (e.g., allogeneic orxenogeneic organ transplantation). With respect to transplantation,compositions of the invention may be administered prior to, concomitantwith, and/or after transplantation. In a specific embodiment,compositions of the invention are administered after transplantation,prior to the beginning of recovery of T-cell populations. In anotherspecific embodiment, compositions of the invention are firstadministered after transplantation after the beginning of recovery of Tcell populations, but prior to full recovery of B cell populations.

[0502] As an agent to boost immunoresponsiveness among individualshaving an acquired loss of B cell function. Conditions resulting in anacquired loss of B cell function that may be ameliorated or treated byadministering the polypeptides, antibodies, polynucleotides and/oragonists or antagonists thereof, include, but are not limited to, HIVInfection, AIDS, bone marrow transplant, and B cell chronic lymphocyticleukemia (CLL).

[0503] As an agent to boost immunoresponsiveness among individualshaving a temporary immune deficiency. Conditions resulting in atemporary immune deficiency that may be ameliorated or treated byadministering the polypeptides, antibodies, polynucleotides and/oragonists or antagonists thereof, include, but are not limited to,recovery from viral infections (e.g., influenza), conditions associatedwith malnutrition, recovery from infectious mononucleosis, or conditionsassociated with stress, recovery from measles, recovery from bloodtransfusion, recovery from surgery.

[0504] As a regulator of antigen presentation by monocytes, dendriticcells, and/or B-cells. In one embodiment, polynucleotides, polypeptides,antibodies, and/or agonists or antagonists of the present inventionenhance antigen presentation or antagonizes antigen presentation invitro or in vivo. Moreover, in related embodiments, said enhancement orantagonization of antigen presentation may be useful as an anti-tumortreatment or to modulate the immune system.

[0505] As an agent to direct an individuals immune system towardsdevelopment of a humoral response (i.e. TH2) as opposed to a TH1cellular response.

[0506] As a means to induce tumor proliferation and thus make it moresusceptible to anti-neoplastic agents. For example, multiple myeloma isa slowly dividing disease and is thus refractory to virtually allanti-neoplastic regimens. If these cells were forced to proliferate morerapidly their susceptibility profile would likely change.

[0507] As a stimulator of B cell production in pathologies such as AIDS,chronic lymphocyte disorder and/or Common Variable Immunodificiency.

[0508] As a therapy for generation and/or regeneration of lymphoidtissues following surgery, trauma or genetic defect.

[0509] As a gene-based therapy for genetically inherited disordersresulting in immuno-incompetence such as observed among SCID patients.

[0510] As an antigen for the generation of antibodies to inhibit orenhance immune mediated responses against polypeptides of the invention.

[0511] As a means of activating T cells.

[0512] As a means of activating monocytes/macrophages to defend againstparasitic diseases that effect monocytes such as Leshmania.

[0513] As pretreatment of bone marrow samples prior to transplant. Suchtreatment would increase B cell representation and thus acceleraterecover.

[0514] As a means of regulating secreted cytokines that are elicited bypolypeptides of the invention.

[0515] Additionally, polypeptides or polynucleotides of the invention,and/or agonists thereof, may be used to treat or prevent IgE-mediatedallergic reactions. Such allergic reactions include, but are not limitedto, asthma, rhinitis, and eczema.

[0516] All of the above described applications as they may apply toveterinary medicine.

[0517] Antagonists of the invention include, for example, binding and/orinhibitory antibodies, antisense nucleic acids, ribozymes or solubleforms of the immunoglobulin-like receptor(s) (e.g., aimmunoglobulin-like-Fc fusion protein) (see e.g., Example 9). Thesewould be expected to reverse many of the activities of the liganddescribed above as well as find clinical or practical application as:

[0518] A means of blocking various aspects of immune responses toforeign agents or self. Examples include autoimmune disorders such aslupus, and arthritis, as well as immunoresponsiveness to skin allergies,inflammation, bowel disease, injury and pathogens.

[0519] A therapy for preventing the B cell proliferation and Igsecretion associated with autoimmune diseases such as idiopathicthrombocytopenic purpura, systemic lupus erythramatosus and MS.

[0520] An inhibitor of B and/or T cell migration in endothelial cells.This activity disrupts tissue architecture or cognate responses and isuseful, for example in disrupting immune responses, and blocking sepsis.

[0521] An inhibitor of graft versus host disease or transplantrejection.

[0522] A therapy for B cell and/or T cell malignancies such as ALL,Hodgkins disease, non-Hodgkins lymphoma, Chronic lymphocyte leukemia,plasmacytomas, multiple myeloma, Burkitt's lymphoma, and EBV-transformeddiseases.

[0523] A therapy for chronic hypergammaglobulinemeia evident in suchdiseases as monoclonalgammopathy of undetermined significance (MGUS),Waldenstrom's disease, related idiopathic monoclonalgammopathies, andplasmacytomas.

[0524] A therapy for decreasing cellular proliferation of Large B-cellLymphomas.

[0525] A means of decreasing the involvement of B cells and Igassociated with Chronic Myelogenous Leukemia.

[0526] An immunosuppressive agent(s).

[0527] Polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention may be used to modulate IgEconcentrations in vitro or in vivo.

[0528] In another embodiment, administration of polypeptides,antibodies, polynucleotides and/or agonists or antagonists of theinvention, may be used to treat or prevent IgE-mediated allergicreactions including, but not limited to, asthma, rhinitis, and eczema.

[0529] The agonists and antagonists may be employed in a compositionwith a pharmaceutically acceptable carrier, e.g., as described herein.

[0530] The agonists or antagonists may be employed for instance toinhibit polypeptide chemotaxis and activation of macrophages and theirprecursors, and of neutrophils, basophils, B lymphocytes and some T-cellsubsets, e.g., activated and CD8 cytotoxic T cells and natural killercells, in certain auto-immune and chronic inflammatory and infectivediseases. Examples of autoimmune diseases are described herein andinclude multiple sclerosis, and insulin-dependent diabetes. Theantagonists or agonists may also be employed to treat infectiousdiseases including silicosis, sarcoidosis, idiopathic pulmonary fibrosisby, for example, preventing the recruitment and activation ofmononuclear phagocytes. They may also be employed to treat idiopathichyper-eosinophilic syndrome by, for example, preventing eosinophilproduction and migration. The antagonists or agonists or may also beemployed for treating atherosclerosis, for example, by preventingmonocyte infiltration in the artery wall.

[0531] Antibodies against polypeptides of the invention may be employedto treat ARDS.

[0532] Agonists and/or antagonists of the invention also have uses instimulating wound and tissue repair, stimulating angiogenesis,stimulating the repair of vascular or lymphatic diseases or disorders.Additionally, agonists and antagonists of the invention may be used tostimulate the regeneration of mucosal surfaces.

[0533] In a specific embodiment, polynucleotides or polypeptides, and/oragonists thereof are used to treat or prevent a disorder characterizedby primary or acquired immunodeficiency, deficient serum immunoglobulinproduction, recurrent infections, and/or immune system dysfunction.Moreover, polynucleotides or polypeptides, and/or agonists thereof maybe used to treat or prevent infections of the joints, bones, skin,and/or parotid glands, blood-borne infections (e.g., sepsis, meningitis,septic arthritis, and/or osteomyelitis), autoimmune diseases (e.g.,those disclosed herein), inflammatory disorders, and malignancies,and/or any disease or disorder or condition associated with theseinfections, diseases, disorders and/or malignancies) including, but notlimited to, CVID, other primary immune deficiencies, HIV disease, CLL,recurrent bronchitis, sinusitis, otitis media, conjunctivitis,pneumonia, hepatitis, meningitis, herpes zoster (e.g., severe herpeszoster), and/or pneumocystis carnii.

[0534] In another embodiment, polynucleotides, polypeptides, antibodies,and/or agonists or antagonists of the present invention are used totreat, and/or diagnose an individual having common variableimmunodeficiency disease (“CVID”; also known as “acquiredagammaglobulinemia” and “acquired hypogammaglobulinemia”) or a subset ofthis disease.

[0535] In a specific embodiment, polynucleotides, polypeptides,antibodies, and/or agonists or antagonists of the present invention maybe used to treat, diagnose, and/or prevent (1) cancers or neoplasms and(2) autoimmune cell or tissue-related cancers or neoplasms. In apreferred embodiment, polynucleotides, polypeptides, antibodies, and/oragonists or antagonists of the present invention conjugated to a toxinor a radioactive isotope, as described herein, may be used to treat,diagnose, and/or prevent acute myelogeneous leukemia. In a furtherpreferred embodiment, polynucleotides, polypeptides, antibodies, and/oragonists or antagonists of the present invention conjugated to a toxinor a radioactive isotope, as described herein, may be used to treat,diagnose, and/or prevent, chronic myelogeneous leukemia, multiplemyeloma, non-Hodgkins lymphoma, and/or Hodgkins disease.

[0536] In another specific embodiment, polynucleotides or polypeptides,and/or agonists or antagonists of the invention may be used to treat,diagnose, prognose, and/or prevent selective IgA deficiency,myeloperoxidase deficiency, C2 deficiency, ataxia-telangiectasia,DiGeorge anomaly, common variable immunodeficiency (CVI), X-linkedagammaglobulinemia, severe combined immunodeficiency (SCID), chronicgranulomatous disease (CGD), and Wiskott-Aldrich syndrome.

[0537] Examples of autoimmune disorders that can be treated or detectedare described above and also include, but are not limited to: Addison'sDisease, hemolytic anemia, antiphospholipid syndrome, rheumatoidarthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis,Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, MyastheniaGravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus,Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome,Autoimmune Thyroiditis, Systemic Lupus Erythematosus, AutoimmunePulmonary Inflammation, Guillain-Barre Syndrome, insulin dependentdiabetes mellitis, and autoimmune inflammatory eye disease.

[0538] In a preferred embodiment, the autoimmune diseases and disordersand/or conditions associated with the diseases and disorders recitedabove are treated, prevented, and/or diagnosed using immunoglobulin-likeantibodies and/or immunoglobulin-like antibodies and/or a solubleimmunoglobulin-like polypeptide of the invention.

[0539] In specific embodiments, the compositions of the invention areused as an agent to boost immunoresponsiveness among B cellimmunodeficient individuals, such as, for example, an individual who hasundergone a partial or complete splenectomy.

[0540] Additionally, polynucleotides, polypeptides, and/or antagonistsof the invention may affect apoptosis, and therefore, would be useful intreating a number of diseases associated with increased cell survival orthe inhibition of apoptosis. For example, diseases associated withincreased cell survival or the inhibition of apoptosis that could betreated or detected by polynucleotides, polypeptides, and/or antagonistsof the invention, include cancers (such as follicular lymphomas,carcinomas with p53 mutations, and hormone-dependent tumors, including,but not limited to colon cancer, cardiac tumors, pancreatic cancer,melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer,testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma,lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi'ssarcoma and ovarian cancer); autoimmune disorders (such as, multiplesclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliarycirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemiclupus erythematosus and immune-related glomerulonephritis and rheumatoidarthritis) and viral infections (such as herpes viruses, pox viruses andadenoviruses), inflammation, graft v. host disease, acute graftrejection, and chronic graft rejection. In preferred embodiments,polynucleotides, polypeptides, and/or antagonists of the invention areused to inhibit growth, progression, and/or metastisis of cancers, inparticular those listed above.

[0541] Additional diseases or conditions associated with increased cellsurvival that could be treated or detected by polynucleotides,polypeptides, and/or antagonists of the invention, include, but are notlimited to, progression, and/or metastases of malignancies and relateddisorders such as leukemia (including acute leukemias (e.g., acutelymphocytic leukemia, acute myelocytic leukemia (including myeloblastic,promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) andchronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia andchronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g.,Hodgkin's disease and non-Hodgkin's disease), multiple myeloma,Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumorsincluding, but not limited to, sarcomas and carcinomas such asfibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

[0542] Diseases associated with increased apoptosis that could betreated or detected by polynucleotides, polypeptides, and/or antagonistsof the invention, include AIDS; neurodegenerative disorders (such asAlzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis,Retinitis pigmentosa, Cerebellar degeneration and brain tumor or priorassociated disease); autoimmune disorders (such as, multiple sclerosis,Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet'sdisease, Crohn's disease, polymyositis, systemic lupus erythematosus andimmune-related glomerulonephritis and rheumatoid arthritis)myelodysplastic syndromes (such as aplastic anemia), graft v. hostdisease, ischemic injury (such as that caused by myocardial infarction,stroke and reperfusion injury), liver injury (e.g., hepatitis relatedliver injury, ischemia/reperfusion injury, cholestosis (bile ductinjury) and liver cancer); toxin-induced liver disease (such as thatcaused by alcohol), septic shock, cachexia and anorexia.

[0543] Hyperproliferative diseases and/or disorders that could bedetected and/or treated by polynucleotides, polypeptides, and/orantagonists of the invention, include, but are not limited to neoplasmslocated in the: liver, abdomen, bone, breast, digestive system,pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary,testicles, ovary, thymus, thyroid), eye, head and neck, nervous (centraland peripheral), lymphatic system, pelvic, skin, soft tissue, spleen,thoracic, and urogenital.

[0544] Similarly, other hyperproliferative disorders can also be treatedor detected by polynucleotides, polypeptides, and/or antagonists of theinvention. Examples of such hyperproliferative disorders include, butare not limited to: hypergammaglobulinemia, lymphoproliferativedisorders, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome,Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, andany other hyperproliferative disease, besides neoplasia, located in anorgan system listed above.

[0545] Hyperproliferative Disorders

[0546] Polynucleotides or polypeptides, or agonists or antagonists ofthe present invention can be used to treat or detect hyperproliferativedisorders, including neoplasms. Polynucleotides or polypeptides, oragonists or antagonists of the present invention may inhibit theproliferation of the disorder through direct or indirect interactions.Alternatively, Polynucleotides or polypeptides, or agonists orantagonists of the present invention may proliferate other cells whichcan inhibit the hyperproliferative disorder.

[0547] For example, by increasing an immune response, particularlyincreasing antigenic qualities of the hyperproliferative disorder or byproliferating, differentiating, or mobilizing T-cells,hyperproliferative disorders can be treated. This immune response may beincreased by either enhancing an existing immune response, or byinitiating a new immune response. Alternatively, decreasing an immuneresponse may also be a method of treating hyperproliferative disorders,such as a chemotherapeutic agent.

[0548] Examples of hyperproliferative disorders that can be treated ordetected by Polynucleotides or polypeptides, or agonists or antagonistsof the present invention include, but are not limited to neoplasmslocated in the: colon, abdomen, bone, breast, digestive system, liver,pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary,testicles, ovary, thymus, thyroid), eye, head and neck, nervous (centraland peripheral), lymphatic system, pelvic, skin, soft tissue, spleen,thoracic, and urogenital.

[0549] Similarly, other hyperproliferative disorders can also be treatedor detected by polynucleotides or polypeptides, or agonists orantagonists of the present invention. Examples of suchhyperproliferative disorders include, but are not limited to:hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias,purpura, sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia,Gaucher's Disease, histiocytosis, and any other hyperproliferativedisease, besides neoplasia, located in an organ system listed above.

[0550] One preferred embodiment utilizes polynucleotides of the presentinvention to inhibit aberrant cellular division, by gene therapy usingthe present invention, and/or protein fusions or fragments thereof.

[0551] Thus, the present invention provides a method for treating cellproliferative disorders by inserting into an abnormally proliferatingcell a polynucleotide of the present invention, wherein saidpolynucleotide represses said expression.

[0552] Another embodiment of the present invention provides a method oftreating cell-proliferative disorders in individuals comprisingadministration of one or more active gene copies of the presentinvention to an abnormally proliferating cell or cells. In a preferredembodiment, polynucleotides of the present invention is a DNA constructcomprising a recombinant expression vector effective in expressing a DNAsequence encoding said polynucleotides. In another preferred embodimentof the present invention, the DNA construct encoding the poynucleotidesof the present invention is inserted into cells to be treated utilizinga retrovirus, or more preferrably an adenoviral vector (See G J. Nabel,et. al., PNAS 1999 96: 324-326, which is hereby incorporated. byreference). In a most preferred embodiment, the viral vector isdefective and will not transform non-proliferating cells, onlyproliferating cells. Moreover, in a preferred embodiment, thepolynucleotides of the present invention inserted into proliferatingcells either alone, or in combination with or fused to otherpolynucleotides, can then be modulated via an external stimulus (i.e.magnetic, specific small molecule, chemical, or drug administration,etc.), which acts upon the promoter upstream of said polynucleotides toinduce expression of the encoded protein product. As such the beneficialtherapeutic affect of the present invention may be expressly modulated(i.e. to increase, decrease, or inhibit expression of the presentinvention) based upon said external stimulus.

[0553] The polynucleotides encoding a polypeptide of the presentinvention may be administered along with other polynucleotides encodingan angiogenic protein. Examples of angiogenic proteins include, but arenot limited to, acidic and basic fibroblast growth factors, VEGF-1,VEGF-2, VEGF-3, epidermal growth factor alpha and beta, platelet-derivedendothelial cell growth factor, platelet-derived growth factor, tumornecrosis factor alpha, hepatocyte growth factor, insulin like growthfactor, colony stimulating factor, macrophage colony stimulating factor,granulocyte/macrophage colony stimulating factor, and nitric oxidesynthase.

[0554] Polynucleotides of the present invention may be useful inrepressing expression of oncogenic genes or antigens. By “repressingexpression of the oncogenic genes” is intended the suppression of thetranscription of the gene, the degradation of the gene transcript(pre-message RNA), the inhibition of splicing, the destruction of themessenger RNA, the prevention of the post-translational modifications ofthe protein, the destruction of the protein, or the inhibition of thenormal function of the protein.

[0555] For local administration to abnormally proliferating cells,polynucleotides of the present invention may be administered by anymethod known to those of skill in the art including, but not limited totransfection, electroporation, microinjection of cells, or in vehiclessuch as liposomes, lipofectin, or as naked polynucleotides, or any othermethod described throughout the specification. The polynucleotide of thepresent invention may be delivered by known gene delivery systems suchas, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845(1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad.Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol.Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yateset al., Nature 313:812 (1985)) known to those skilled in the art. Thesereferences are exemplary only and are hereby incorporated by reference.In order to specifically deliver or transfect cells which are abnormallyproliferating and spare non-dividing cells, it is preferable to utilizea retrovirus, or adenoviral (as described in the art and elsewhereherein) delivery system known to those of skill in the art. Since hostDNA replication is required for retroviral DNA to integrate and theretrovirus will be unable to self replicate due to the lack of theretrovirus genes needed for its life cycle. Utilizing such a retroviraldelivery system for polynucleotides of the present invention will targetsaid gene and constructs to abnormally proliferating cells and willspare the non-dividing normal cells.

[0556] The polynucleotides of the present invention may be delivereddirectly to cell proliferative disorder/disease sites in internalorgans, body cavities and the like by use of imaging devices used toguide an injecting needle directly to the disease site. Thepolynucleotides of the present invention may also be administered todisease sites at the time of surgical intervention.

[0557] By “cell proliferative disease” is meant any human or animaldisease or disorder, affecting any one or any combination of organs,cavities, or body parts, which is characterized by single or multiplelocal abnormal proliferations of cells, groups of cells, or tissues,whether benign or malignant.

[0558] Any amount of the polynucleotides of the present invention may beadministered as long as it has a biologically inhibiting effect on theproliferation of the treated cells. Moreover, it is possible toadminister more than one of the polynucleotide of the present inventionsimultaneously to the same site. By “biologically inhibiting” is meantpartial or total growth inhibition as well as decreases in the rate ofproliferation or growth of the cells. The biologically inhibitory dosemay be determined by assessing the effects of the polynucleotides of thepresent invention on target malignant or abnormally proliferating cellgrowth in tissue culture, tumor growth in animals and cell cultures, orany other method known to one of ordinary skill in the art.

[0559] The present invention is further directed to antibody-basedtherapies which involve administering of anti-polypeptides andanti-polynucleotide antibodies to a mammalian, preferably human, patientfor treating one or more of the described disorders. Methods forproducing anti-polypeptides and anti-polynucleotide antibodiespolyclonal and monoclonal antibodies are described in detail elsewhereherein. Such antibodies may be provided in pharmaceutically acceptablecompositions as known in the art or as described herein.

[0560] A summary of the ways in which the antibodies of the presentinvention may be used therapeutically includes binding polynucleotidesor polypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

[0561] In particular, the antibodies, fragments and derivatives of thepresent invention are useful for treating a subject having or developingcell proliferative and/or differentiation disorders as described herein.Such treatment comprises administering a single or multiple doses of theantibody, or a fragment, derivative, or a conjugate thereof.

[0562] The antibodies of this invention may be advantageously utilizedin combination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors, for example., which serveto increase the number or activity of effector cells which interact withthe antibodies.

[0563] It is preferred to use high affinity and/or potent in vivoinhibiting and/or neutralizing antibodies against polypeptides orpolynucleotides of the present invention, fragments or regions thereof,for both immunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragements thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides,including fragements thereof. Preferred binding affinities include thosewith a dissociation constant or Kd less than 5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁷M,10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M, 5×10⁻¹⁰M, 10⁻¹⁰M, 5×10⁻¹¹M,10⁻¹¹M, 5×10⁻¹²M, 10⁻¹²M, ×10⁻¹³M, 10⁻¹³M, 5×10⁻¹⁴M, 10⁻¹⁴M, 5×10⁻¹⁵M,and 10⁻¹⁵M.

[0564] Moreover, polypeptides of the present invention are useful ininhibiting the angiogenesis of proliferative cells or tissues, eitheralone, as a protein fusion, or in combination with other polypeptidesdirectly or indirectly, as described elsewhere herein. In a mostpreferred embodiment, said anti-angiogenesis effect may be achievedindirectly, for example, through the inhibition of hematopoietic,tumor-specific cells, such as tumor-associated macrophages (See Joseph IB, et al. J Natl Cancer Inst, 90(21):1648-53 (1998), which is herebyincorporated by reference). Antibodies directed to polypeptides orpolynucleotides of the present invention may also result in inhibitionof angiogenesis directly, or indirectly (See Witte L, et al., CancerMetastasis Rev. 17(2):155-61 (1998), which is hereby incorporated byreference)).

[0565] Polypeptides, including protein fusions, of the presentinvention, or fragments thereof may be useful in inhibitingproliferative cells or tissues through the induction of apoptosis. Saidpolypeptides may act either directly, or indirectly to induce apoptosisof proliferative cells and tissues, for example in the activation of adeath-domain receptor, such as tumor necrosis factor (TNF) receptor-1,CD95 (Fas/APO-1), TNF-receptor-related apoptosis-mediated protein(TRAMP) and TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and2 (See Schulze-Osthoff K, et.al., Eur J Biochem 254(3):439-59 (1998),which is hereby incorporated by reference). Moreover, in anotherpreferred embodiment of the present invention, said polypeptides mayinduce apoptosis through other mechanisms, such as in the activation ofother proteins which will activate apoptosis, or through stimulating theexpression of said proteins, either alone or in combination with smallmolecule drugs or adjuviants, such as apoptonin, galectins,thioredoxins, antiinflammatory proteins (See for example, Mutat Res400(1-2):447-55 (1998), Med Hypotheses.50(5):423-33 (1998) BiolInteract. Apr 24;111-112:23-34 (1998), J Mol Med.76(6):402-12 (1998),Int J Tissue React;20(1):3-15 (1998), which are all hereby incorporatedby reference).

[0566] Polypeptides, including protein fusions to, or fragments thereof,of the present invention are useful in inhibiting the metastasis ofproliferative cells or tissues. Inhibition may occur as a direct resultof administering polypeptides, or antibodies directed to saidpolypeptides as described elsewere herein, or indirectly, such asactivating the expression of proteins known to inhibit metastasis, forexample alpha 4 integrins, (See, e.g., Curr Top Microbiol Immunol1998;231:125-41, which is hereby incorporated by reference). Suchthereapeutic affects of the present invention may be achieved eitheralone, or in combination with small molecule drugs or adjuvants.

[0567] In another embodiment, the invention provides a method ofdelivering compositions containing the polypeptides of the invention(e.g., compositions containing polypeptides or polypeptide antibodesassociated with heterologous polypeptides, heterologous nucleic acids,toxins, or prodrugs) to targeted cells expressing the polypeptide of thepresent invention. Polypeptides or polypeptide antibodes of theinvention may be associated with with heterologous polypeptides,heterologous nucleic acids, toxins, or prodrugs via hydrophobic,hydrophilic, ionic and/or covalent interactions.

[0568] Polypeptides, protein fusions to, or fragments thereof, of thepresent invention are useful in enhancing the immunogenicity and/orantigenicity of proliferating cells or tissues, either directly, such aswould occur if the polypeptides of the present invention ‘vaccinated’the immune response to respond to proliferative antigens and immunogens,or indirectly, such as in activating the expression of proteins known toenhance the immune response (e.g. chemokines), to said antigens andimmunogens.

[0569] Cardiovascular Disorders

[0570] Polynucleotides or polypeptides, or agonists or antagonists ofthe present invention, may be used to treat cardiovascular disorders,including peripheral artery disease, such as limb ischemia.

[0571] Cardiovascular disorders include cardiovascular abnormalities,such as arterio-arterial fistula, arteriovenous fistula, cerebralarteriovenous malformations, congenital heart defects, pulmonaryatresia, and Scimitar Syndrome. Congenital heart defects include aorticcoarctation, cor triatriatum, coronary vessel anomalies, crisscrossheart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly,Eisenmenger complex, hypoplastic left heart syndrome, levocardia,tetralogy of fallot, transposition of great vessels, double outlet rightventricle, tricuspid atresia, persistent truncus arteriosus, and heartseptal defects, such as aortopulmonary septal defect, endocardialcushion defects, Lutembacher's Syndrome, trilogy of Fallot, ventricularheart septal defects.

[0572] Cardiovascular disorders also include heart disease, such asarrhythmias, carcinoid heart disease, high cardiac output, low cardiacoutput, cardiac tamponade, endocarditis (including bacterial), heartaneurysm, cardiac arrest, congestive heart failure, congestivecardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy,congestive cardiomyopathy, left ventricular hypertrophy, rightventricular hypertrophy, post-infarction heart rupture, ventricularseptal rupture, heart valve diseases, myocardial diseases, myocardialischemia, pericardial effusion, pericarditis (including constrictive andtuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonaryheart disease, rheumatic heart disease, ventricular dysfunction,hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome,cardiovascular syphilis, and cardiovascular tuberculosis.

[0573] Arrhythmias include sinus arrhythmia, atrial fibrillation, atrialflutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branchblock, sinoatrial block, long QT syndrome, parasystole,Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome,Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, andventricular fibrillation. Tachycardias include paroxysmal tachycardia,supraventricular tachycardia, accelerated idioventricular rhythm,atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia,ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia,sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

[0574] Heart valve disease include aortic valve insufficiency, aorticvalve stenosis, hear murmurs, aortic valve prolapse, mitral valveprolapse, tricuspid valve prolapse, mitral valve insufficiency, mitralvalve stenosis, pulmonary atresia, pulmonary valve insufficiency,pulmonary valve stenosis, tricuspid atresia, tricuspid valveinsufficiency, and tricuspid valve stenosis.

[0575] Myocardial diseases include alcoholic cardiomyopathy, congestivecardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvularstenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy,Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardialfibrosis, Kearns Syndrome, myocardial reperfusion injury, andmyocarditis.

[0576] Myocardial ischemias include coronary disease, such as anginapectoris, coronary aneurysm, coronary arteriosclerosis, coronarythrombosis, coronary vasospasm, myocardial infarction and myocardialstunning.

[0577] Cardiovascular diseases also include vascular diseases such asaneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-WeberSyndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis,aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis,enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabeticangiopathies, diabetic retinopathy, embolisms, thrombosis,erythromelalgia, hemorrhoids, hepatic veno-occlusive disease,hypertension, hypotension, ischemia, peripheral vascular diseases,phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CRESTsyndrome, retinal vein occlusion, Scimitar syndrome, superior vena cavasyndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagictelangiectasia, yaricocele, varicose veins, varicose ulcer, vasculitis,and venous insufficiency.

[0578] Aneurysms include dissecting aneurysms, false aneurysms, infectedaneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms,coronary aneurysms, heart aneurysms, and iliac aneurysms.

[0579] Arterial occlusive diseases include arteriosclerosis,intermittent claudication, carotid stenosis, fibromuscular dysplasias,mesenteric vascular occlusion, Moyamoya disease, renal arteryobstruction, retinal artery occlusion, and thromboangiitis obliterans.

[0580] Cerebrovascular disorders include carotid artery diseases,cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia,cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebralartery diseases, cerebral embolism and thrombosis, carotid arterythrombosis, sinus thrombosis, Wallenberg's syndrome, cerebralhemorrhage, epidural hematoma, subdural hematoma, subaraxhnoidhemorrhage, cerebral infarction, cerebral ischemia (includingtransient), subclavian steal syndrome, periventricular leukomalacia,vascular headache, cluster headache, migraine, and vertebrobasilarinsufficiency.

[0581] Embolisms include air embolisms, amniotic fluid embolisms,cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonaryembolisms, and thromoboembolisms. Thrombosis include coronarythrombosis, hepatic vein thrombosis, retinal vein occlusion, carotidartery thrombosis, sinus thrombosis, Wallenberg's syndrome, andthrombophlebitis.

[0582] Ischemia includes cerebral ischemia, ischemic colitis,compartment syndromes, anterior compartment syndrome, myocardialischemia, reperfusion injuries, and peripheral limb ischemia. Vasculitisincludes aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome,mucocutaneous lymph node syndrome, thromboangiitis obliterans,hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergiccutaneous vasculitis, and Wegener's granulomatosis.

[0583] Polynucleotides or polypeptides, or agonists or antagonists ofthe present invention, are especially effective for the treatment ofcritical limb ischemia and coronary disease.

[0584] Polypeptides may be administered using any method known in theart, including, but not limited to, direct needle injection at thedelivery site, intravenous injection, topical administration, catheterinfusion, biolistic injectors, particle accelerators, gelfoam spongedepots, other commercially available depot materials, osmotic pumps,oral or suppositorial solid pharmaceutical formulations, decanting ortopical applications during surgery, aerosol delivery. Such methods areknown in the art. Polypeptides may be administered as part of aTherapeutic, described in more detail below. Methods of deliveringpolynucleotides are described in more detail herein.

[0585] Anti-Angiogenesis Activity

[0586] The naturally occurring balance between endogenous stimulatorsand inhibitors of angiogenesis is one in which inhibitory influencespredominate. Rastinejad et al., Cell 56:345-355 (1989). In those rareinstances in which neovascularization occurs under normal physiologicalconditions, such as wound healing, organ regeneration, embryonicdevelopment, and female reproductive processes, angiogenesis isstringently regulated and spatially and temporally delimited. Underconditions of pathological angiogenesis such as that characterizingsolid tumor growth, these regulatory controls fail. Unregulatedangiogenesis becomes pathologic and sustains progression of manyneoplastic and non-neoplastic diseases. A number of serious diseases aredominated by abnormal neovascularization including solid tumor growthand metastases, arthritis, some types of eye disorders, and psoriasis.See, e.g., reviews by Moses et al., Biotech. 9:630-634 (1991); Folkmanet al, N. Engl. J. Med., 333:1757-1763 (1995); Auerbach et al., J.Microvasc. Res. 29:401-411 (1985); Folkman, Advances in Cancer Research,eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985);Patz, Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al., Science221:719-725 (1983). In a number of pathological conditions, the processof angiogenesis contributes to the disease state. For example,significant data have accumulated which suggest that the growth of solidtumors is dependent on angiogenesis. Folkman and Klagsbrun, Science235:442-447 (1987).

[0587] The present invention provides for treatment of diseases ordisorders associated with neovascularization by administration of thepolynucleotides and/or polypeptides of the invention, as well asagonists or antagonists of the present invention. Malignant andmetastatic conditions which can be treated with the polynucleotides andpolypeptides, or agonists or antagonists of the invention include, butare not limited to, malignancies, solid tumors, and cancers describedherein and otherwise known in the art (for a review of such disorders,see Fishman et al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia(1985)).Thus, the present invention provides a method of treating anangiogenesis-related disease and/or disorder, comprising administeringto an individual in need thereof a therapeutically effective amount of apolynucleotide, polypeptide, antagonist and/or agonist of the invention.For example, polynucleotides, polypeptides, antagonists and/or agonistsmay be utilized in a variety of additional methods in order totherapeutically treat a cancer or tumor. Cancers which may be treatedwith polynucleotides, polypeptides, antagonists and/or agonists include,but are not limited to solid tumors, including prostate, lung, breast,ovarian, stomach, pancreas, larynx, esophagus, testes, liver, parotid,biliary tract, colon, rectum, cervix, uterus, endometrium, kidney,bladder, thyroid cancer; primary tumors and metastases; melanomas;glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non- small cell lungcancer; colorectal cancer; advanced malignancies; and blood born tumorssuch as leukemias. For example, polynucleotides, polypeptides,antagonists and/or agonists may be delivered topically, in order totreat cancers such as skin cancer, head and neck tumors, breast tumors,and Kaposi's sarcoma.

[0588] Within yet other aspects, polynucleotides, polypeptides,antagonists and/or agonists may be utilized to treat superficial formsof bladder cancer by, for example, intravesical administration.Polynucleotides, polypeptides, antagonists and/or agonists may bedelivered directly into the tumor, or near the tumor site, via injectionor a catheter. Of course, as the artisan of ordinary skill willappreciate, the appropriate mode of administration will vary accordingto the cancer to be treated. Other modes of delivery are discussedherein.

[0589] Polynucleotides, polypeptides, antagonists and/or agonists may beuseful in treating other disorders, besides cancers, which involveangiogenesis. These disorders include, but are not limited to: benigntumors, for example hemangiomas, acoustic neuromas, neurofibromas,trachomas, and pyogenic granulomas; artheroscleric plaques; ocularangiogenic diseases, for example, diabetic retinopathy, retinopathy ofprematurity, macular degeneration; corneal graft rejection, neovascularglaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis andPterygia (abnormal blood vessel growth) of the eye; rheumatoidarthritis; psoriasis; delayed wound healing; endometriosis;vasculogenesis; granulations; hypertrophic scars (keloids); nonunionfractures; scleroderma; trachoma; vascular adhesions; myocardialangiogenesis; coronary collaterals; cerebral collaterals; arteriovenousmalformations; ischemic limb angiogenesis; Osler-Webber Syndrome; plaqueneovascularization; telangiectasia; hemophiliac joints; angiofibroma;fibromuscular dysplasia; wound granulation; Crohn's disease; andatherosclerosis.

[0590] For example, within one aspect of the present invention methodsare provided for treating hypertrophic scars and keloids, comprising thestep of administering a polynucleotide, polypeptide, antagonist and/oragonist of the invention to a hypertrophic scar or keloid.

[0591] Within one embodiment of the present invention polynucleotides,polypeptides, antagonists and/or agonists are directly injected into ahypertrophic scar or keloid, in order to prevent the progression ofthese lesions. This therapy is of particular value in the prophylactictreatment of conditions which are known to result in the development ofhypertrophic scars and keloids (e.g., burns), and is preferablyinitiated after the proliferative phase has had time to progress(approximately 14 days after the initial injury), but beforehypertrophic scar or keloid development. As noted above, the presentinvention also provides methods for treating neovascular diseases of theeye, including for example, corneal neovascularization, neovascularglaucoma, proliferative diabetic retinopathy, retrolental fibroplasiaand macular degeneration.

[0592] Moreover, Ocular disorders associated with neovascularizationwhich can be treated with the polynucleotides and polypeptides of thepresent invention (including agonists and/or antagonists) include, butare not limited to: neovascular glaucoma, diabetic retinopathy,retinoblastoma, retrolental fibroplasia, uveitis, retinopathy ofprematurity macular degeneration, corneal graft neovascularization, aswell as other eye inflammatory diseases, ocular tumors and diseasesassociated with choroidal or iris neovascularization. See, e.g., reviewsby Waltman et al., Am. J. Ophthal. 85:704-710 (1978) and Gartner et al.,Surv. Ophthal. 22:291-312 (1978).

[0593] Thus, within one aspect of the present invention methods areprovided for treating neovascular diseases of the eye such as cornealneovascularization (including corneal graft neovascularization),comprising the step of administering to a patient a therapeuticallyeffective amount of a compound (as described above) to the cornea, suchthat the formation of blood vessels is inhibited. Briefly, the cornea isa tissue which normally lacks blood vessels. In certain pathologicalconditions however, capillaries may extend into the cornea from thepericorneal vascular plexus of the limbus. When the cornea becomesvascularized, it also becomes clouded, resulting in a decline in thepatient's visual acuity. Visual loss may become complete if the corneacompletely opacitates. A wide variety of disorders can result in cornealneovascularization, including for example, corneal infections (e.g.,trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis),immunological processes (e.g., graft rejection and Stevens-Johnson'ssyndrome), alkali burns, trauma, inflammation (of any cause), toxic andnutritional deficiency states, and as a complication of wearing contactlenses.

[0594] Within particularly preferred embodiments of the invention, maybe prepared for topical administration in saline (combined with any ofthe preservatives and antimicrobial agents commonly used in ocularpreparations), and administered in eyedrop form. The solution orsuspension may be prepared in its pure form and administered severaltimes daily. Alternatively, anti-angiogenic compositions, prepared asdescribed above, may also be administered directly to the cornea. Withinpreferred embodiments, the anti-angiogenic composition is prepared witha muco-adhesive polymer which binds to cornea. Within furtherembodiments, the anti-angiogenic factors or anti-angiogenic compositionsmay be utilized as an adjunct to conventional steroid therapy. Topicaltherapy may also be useful prophylactically in corneal lesions which areknown to have a high probability of inducing an angiogenic response(such as chemical burns). In these instances the treatment, likely incombination with steroids, may be instituted immediately to help preventsubsequent complications.

[0595] Within other embodiments, the compounds described above may beinjected directly into the corneal stroma by an ophthalmologist undermicroscopic guidance. The preferred site of injection may vary with themorphology of the individual lesion, but the goal of the administrationwould be to place the composition at the advancing front of thevasculature (i.e., interspersed between the blood vessels and the normalcornea). In most cases this would involve perilimbic corneal injectionto “protect” the cornea from the advancing blood vessels. This methodmay also be utilized shortly after a corneal insult in order toprophylactically prevent corneal neovascularization. In this situationthe material could be injected in the perilimbic cornea interspersedbetween the corneal lesion and its undesired potential limbic bloodsupply. Such methods may also be utilized in a similar fashion toprevent capillary invasion of transplanted corneas. In asustained-release form injections might only be required 2-3 times peryear. A steroid could also be added to the injection solution to reduceinflammation resulting from the injection itself.

[0596] Within another aspect of the present invention, methods areprovided for treating neovascular glaucoma, comprising the step ofadministering to a patient a therapeutically effective amount of apolynucleotide, polypeptide, antagonist and/or agonist to the eye, suchthat the formation of blood vessels is inhibited. In one embodiment, thecompound may be administered topically to the eye in order to treatearly forms of neovascular glaucoma. Within other embodiments, thecompound may be implanted by injection into the region of the anteriorchamber angle. Within other embodiments, the compound may also be placedin any location such that the compound is continuously released into theaqueous humor. Within another aspect of the present invention, methodsare provided for treating proliferative diabetic retinopathy, comprisingthe step of administering to a patient a therapeutically effectiveamount of a polynucleotide, polypeptide, antagonist and/or agonist tothe eyes, such that the formation of blood vessels is inhibited.

[0597] Within particularly preferred embodiments of the invention,proliferative diabetic retinopathy may be treated by injection into theaqueous humor or the vitreous, in order to increase the localconcentration of the polynucleotide, polypeptide, antagonist and/oragonist in the retina. Preferably, this treatment should be initiatedprior to the acquisition of severe disease requiring photocoagulation.

[0598] Within another aspect of the present invention, methods areprovided for treating retrolental fibroplasia, comprising the step ofadministering to a patient a therapeutically effective amount of apolynucleotide, polypeptide, antagonist and/or agonist to the eye, suchthat the formation of blood vessels is inhibited. The compound may beadministered topically, via intravitreous injection and/or viaintraocular implants.

[0599] Additionally, disorders which can be treated with thepolynucleotides, polypeptides, agonists and/or agonists include, but arenot limited to, hemangioma, arthritis, psoriasis, angiofibroma,atherosclerotic plaques, delayed wound healing, granulations, hemophilicjoints, hypertrophic scars, nonunion fractures, Osler-Weber syndrome,pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.

[0600] Moreover, disorders and/or states, which can be treated with betreated with the the polynucleotides, polypeptides, agonists and/oragonists include, but are not limited to, solid tumors, blood borntumors such as leukemias, tumor metastasis, Kaposi's sarcoma, benigntumors, for example hemangiomas, acoustic neuromas, neurofibromas,trachomas, and pyogenic granulomas, rheumatoid arthritis, psoriasis,ocular angiogenic diseases, for example, diabetic retinopathy,retinopathy of prematurity, macular degeneration, corneal graftrejection, neovascular glaucoma, retrolental fibroplasia, rubeosis,retinoblastoma, and uvietis, delayed wound healing, endometriosis,vascluogenesis, granulations, hypertrophic scars (keloids), nonunionfractures, scleroderma, trachoma, vascular adhesions, myocardialangiogenesis, coronary collaterals, cerebral collaterals, arteriovenousmalformations, ischemic limb angiogenesis, Osler-Webber Syndrome, plaqueneovascularization, telangiectasia, hemophiliac joints, angiofibromafibromuscular dysplasia, wound granulation, Crohn's disease,atherosclerosis, birth control agent by preventing vascularizationrequired for embryo implantation controlling menstruation, diseases thathave angiogenesis as a pathologic consequence such as cat scratchdisease (Rochele minalia quintosa), ulcers (Helicobacter pylori),Bartonellosis and bacillary angiomatosis.

[0601] In one aspect of the birth control method, an amount of thecompound sufficient to block embryo implantation is administered beforeor after intercourse and fertilization have occurred, thus providing aneffective method of birth control, possibly a “morning after” method.Polynucleotides, polypeptides, agonists and/or agonists may also be usedin controlling menstruation or administered as either a peritoneallavage fluid or for peritoneal implantation in the treatment ofendometriosis.

[0602] Polynucleotides, polypeptides, agonists and/or agonists of thepresent invention may be incorporated into surgical sutures in order toprevent stitch granulomas.

[0603] Polynucleotides, polypeptides, agonists and/or agonists may beutilized in a wide variety of surgical procedures. For example, withinone aspect of the present invention a compositions (in the form of, forexample, a spray or film) may be utilized to coat or spray an area priorto removal of a tumor, in order to isolate normal surrounding tissuesfrom malignant tissue, and/or to prevent the spread of disease tosurrounding tissues. Within other aspects of the present invention,compositions (e.g., in the form of a spray) may be delivered viaendoscopic procedures in order to coat tumors, or inhibit angiogenesisin a desired locale.

[0604] Within yet other aspects of the present invention, surgicalmeshes which have been coated with anti-angiogenic compositions of thepresent invention may be utilized in any procedure wherein a surgicalmesh might be utilized. For example, within one embodiment of theinvention a surgical mesh laden with an anti-angiogenic composition maybe utilized during abdominal cancer resection surgery (e.g., subsequentto colon resection) in order to provide support to the structure, and torelease an amount of the anti-angiogenic factor.

[0605] Within further aspects of the present invention, methods areprovided for treating tumor excision sites, comprising administering apolynucleotide, polypeptide, agonist and/or agonist to the resectionmargins of a tumor subsequent to excision, such that the localrecurrence of cancer and the formation of new blood vessels at the siteis inhibited. Within one embodiment of the invention, theanti-angiogenic compound is administered directly to the tumor excisionsite (e.g., applied by swabbing, brushing or otherwise coating theresection margins of the tumor with the anti-angiogenic compound).Alternatively, the anti-angiogenic compounds may be incorporated intoknown surgical pastes prior to administration. Within particularlypreferred embodiments of the invention, the anti-angiogenic compoundsare applied after hepatic resections for malignancy, and afterneurosurgical operations.

[0606] Within one aspect of the present invention, polynucleotides,polypeptides, agonists and/or agonists may be administered to theresection margin of a wide variety of tumors, including for example,breast, colon, brain and hepatic tumors. For example, within oneembodiment of the invention, anti-angiogenic compounds may beadministered to the site of a neurological tumor subsequent to excision,such that the formation of new blood vessels at the site are inhibited.

[0607] The polynucleotides, polypeptides, agonists and/or agonists ofthe present invention may also be administered along with otheranti-angiogenic factors. Representative examples of otheranti-angiogenic factors include: Anti-Invasive Factor, retinoic acid andderivatives thereof, paclitaxel, Suramin, Tissue Inhibitor ofMetalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2,Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2,and various forms of the lighter “d group” transition metals.

[0608] Lighter “d group” transition metals include, for example,vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species.Such transition metal species may form transition metal complexes.Suitable complexes of the above-mentioned transition metal speciesinclude oxo transition metal complexes.

[0609] Representative examples of vanadium complexes include oxovanadium complexes such as vanadate and vanadyl complexes. Suitablevanadate complexes include metavanadate and orthovanadate complexes suchas, for example, ammonium metavanadate, sodium metavanadate, and sodiumorthovanadate. Suitable vanadyl complexes include, for example, vanadylacetylacetonate and vanadyl sulfate including vanadyl sulfate hydratessuch as vanadyl sulfate mono- and trihydrates.

[0610] Representative examples of tungsten and molybdenum complexes alsoinclude oxo complexes. Suitable oxo tungsten complexes include tungstateand tungsten oxide complexes. Suitable tungstate complexes includeammonium tungstate, calcium tungstate, sodium tungstate dihydrate, andtungstic acid. Suitable tungsten oxides include tungsten (IV) oxide andtungsten (VI) oxide. Suitable oxo molybdenum complexes includemolybdate, molybdenum oxide, and molybdenyl complexes. Suitablemolybdate complexes include ammonium molybdate and its hydrates, sodiummolybdate and its hydrates, and potassium molybdate and its hydrates.Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum(VI) oxide, and molybdic acid. Suitable molybdenyl complexes include,for example, molybdenyl acetylacetonate. Other suitable tungsten andmolybdenum complexes include hydroxo derivatives derived from, forexample, glycerol, tartaric acid, and sugars.

[0611] A wide variety of other anti-angiogenic factors may also beutilized within the context of the present invention. Representativeexamples include platelet factor 4; protamine sulphate; sulphated chitinderivatives (prepared from queen crab shells), (Murata et al., CancerRes. 51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex(SP-PG) (the function of this compound may be enhanced by the presenceof steroids such as estrogen, and tamoxifen citrate); Staurosporine;modulators of matrix metabolism, including for example, proline analogs,cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline,alpha,alpha-dipyridyl, aminopropionitrile fumarate;4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone;Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J.Bio. Chem. 267:17321-17326, 1992); Chymostatin (Tomkinson et al.,Biochem J. 286:475-480, 1992); Cyclodextrin Tetradecasulfate;Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557,1990); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin.Invest. 79:1440-1446, 1987); anticollagenase-serum; alpha2-antiplasmin(Holmes et al., J. Biol. Chem. 262(4):1659-1664, 1987); Bisantrene(National Cancer Institute); Lobenzarit disodium(N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”;Takeuchi et al., Agents Actions 36:312-316, 1992); Thalidomide;Angostatic steroid; AGM-1470; carboxynaminolmidazole; andmetalloproteinase inhibitors such as BB94.

[0612] Diseases at the Cellular Level

[0613] Diseases associated with increased cell survival or theinhibition of apoptosis that could be treated or detected bypolynucleotides or polypeptides, as well as antagonists or agonists ofthe present invention, include cancers (such as follicular lymphomas,carcinomas with p53 mutations, and hormone-dependent tumors, including,but not limited to colon cancer, cardiac tumors, pancreatic cancer,melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer,testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma,lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi'ssarcoma and ovarian cancer); autoimmune disorders (such as, multiplesclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliarycirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemiclupus erythematosus and immune-related glomerulonephritis and rheumatoidarthritis) and viral infections (such as herpes viruses, pox viruses andadenoviruses), inflammation, graft v. host disease, acute graftrejection, and chronic graft rejection. In preferred embodiments,polynucleotides, polypeptides, and/or antagonists of the invention areused to inhibit growth, progression, and/or metasis of cancers, inparticular those listed above.

[0614] Additional diseases or conditions associated with increased cellsurvival that could be treated or detected by polynucleotides orpolypeptides, or agonists or antagonists of the present inventioninclude, but are not limited to, progression, and/or metastases ofmalignancies and related disorders such as leukemia (including acuteleukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia(including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, and retinoblastoma.

[0615] Diseases associated with increased apoptosis that could betreated or detected by polynucleotides or polypeptides, as well asagonists or antagonists of the present invention, include AIDS;neurodegenerative disorders (such as Alzheimer's disease, Parkinson'sdisease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellardegeneration and brain tumor or prior associated disease); autoimmunedisorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto'sthyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease,polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes(such as aplastic anemia), graft v. host disease, ischemic injury (suchas that caused by myocardial infarction, stroke and reperfusion injury),liver injury (e.g., hepatitis related liver injury, ischemia/reperfusioninjury, cholestosis (bile duct injury) and liver cancer); toxin-inducedliver disease (such as that caused by alcohol), septic shock, cachexiaand anorexia.

[0616] Wound Healing and Epithelial Cell Proliferation

[0617] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing polynucleotides orpolypeptides, as well as agonists or antagonists of the presentinvention, for therapeutic purposes, for example, to stimulateepithelial cell proliferation and basal keratinocytes for the purpose ofwound healing, and to stimulate hair follicle production and healing ofdermal wounds. Polynucleotides or polypeptides, as well as agonists orantagonists of the present invention, may be clinically useful instimulating wound healing including surgical wounds, excisional wounds,deep wounds involving damage of the dermis and epidermis, eye tissuewounds, dental tissue wounds, oral cavity wounds, diabetic ulcers,dermal ulcers, cubitus ulcers, arterial ulcers, venous stasis ulcers,burns resulting from heat exposure or chemicals, and other abnormalwound healing conditions such as uremia, malnutrition, vitamindeficiencies and complications associted with systemic treatment withsteroids, radiation therapy and antineoplastic drugs andantimetabolites. Polynucleotides or polypeptides, as well as agonists orantagonists of the present invention, could be used to promote dermalreestablishment subsequent to dermal loss

[0618] Polynucleotides or polypeptides, as well as agonists orantagonists of the present invention, could be used to increase theadherence of skin grafts to a wound bed and to stimulatere-epithelialization from the wound bed. The following are types ofgrafts that polynucleotides or polypeptides, agonists or antagonists ofthe present invention, could be used to increase adherence to a woundbed: autografts, artificial skin, allografts, autodermic graft,autoepdermic grafts, avacular grafts, Blair-Brown grafts, bone graft,brephoplastic grafts, cutis graft, delayed graft, dermic graft,epidermic graft, fascia graft, full thickness graft, heterologous graft,xenograft, homologous graft, hyperplastic graft, lamellar graft, meshgraft, mucosal graft, Ollier-Thiersch graft, omenpal graft, patch graft,pedicle graft, penetrating graft, split skin graft, thick split graft.Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention, can be used to promote skin strength and toimprove the appearance of aged skin.

[0619] It is believed that polynucleotides or polypeptides, as well asagonists or antagonists of the present invention, will also producechanges in hepatocyte proliferation, and epithelial cell proliferationin the lung, breast, pancreas, stomach, small intesting, and largeintestine. Polynucleotides or polypeptides, as well as agonists orantagonists of the present invention, could promote proliferation ofepithelial cells such as sebocytes, hair follicles, hepatocytes, type IIpneumocytes, mucin-producing goblet cells, and other epithelial cellsand their progenitors contained within the skin, lung, liver, andgastrointestinal tract. Polynucleotides or polypeptides, agonists orantagonists of the present invention, may promote proliferation ofendothelial cells, keratinocytes, and basal keratinocytes.

[0620] Polynucleotides or polypeptides, as well as agonists orantagonists of the present invention, could also be used to reduce theside effects of gut toxicity that result from radiation, chemotherapytreatments or viral infections. Polynucleotides or polypeptides, as wellas agonists or antagonists of the present invention, may have acytoprotective effect on the small intestine mucosa. Polynucleotides orpolypeptides, as well as agonists or antagonists of the presentinvention, may also stimulate healing of mucositis (mouth ulcers) thatresult from chemotherapy and viral infections.

[0621] Polynucleotides or polypeptides, as well as agonists orantagonists of the present invention, could further be used in fallregeneration of skin in full and partial thickness skin defects,including burns, (i.e., repopulation of hair follicles, sweat glands,and sebaceous glands), treatment of other skin defects such aspsoriasis. Polynucleotides or polypeptides, as well as agonists orantagonists of the present invention, could be used to treatepidermolysis bullosa, a defect in adherence of the epidermis to theunderlying dermis which results in frequent, open and painful blistersby accelerating reepithelialization of these lesions. Polynucleotides orpolypeptides, as well as agonists or antagonists of the presentinvention, could also be used to treat gastric and doudenal ulcers andhelp heal by scar formation of the mucosal lining and regeneration ofglandular mucosa and duodenal mucosal lining more rapidly. Inflamamatorybowel diseases, such as Crohn's disease and ulcerative colitis, arediseases which result in destruction of the mucosal surface of the smallor large intestine, respectively. Thus, polynucleotides or polypeptides,as well as agonists or antagonists of the present invention, could beused to promote the resurfacing of the mucosal surface to aid more rapidhealing and to prevent progression of inflammatory bowel disease.Treatment with polynucleotides or polypeptides, agonists or antagonistsof the present invention, is expected to have a significant effect onthe production of mucus throughout the gastrointestinal tract and couldbe used to protect the intestinal mucosa from injurious substances thatare ingested or following surgery. Polynucleotides or polypeptides, aswell as agonists or antagonists of the present invention, could be usedto treat diseases associate with the under expression.

[0622] Moreover, polynucleotides or polypeptides, as well as agonists orantagonists of the present invention, could be used to prevent and healdamage to the lungs due to various pathological states. Polynucleotidesor polypeptides, as well as agonists or antagonists of the presentinvention, which could stimulate proliferation and differentiation andpromote the repair of alveoli and brochiolar epithelium to prevent ortreat acute or chronic lung damage. For example, emphysema, whichresults in the progressive loss of aveoli, and inhalation injuries,i.e., resulting from smoke inhalation and burns, that cause necrosis ofthe bronchiolar epithelium and alveoli could be effectively treatedusing polynucleotides or polypeptides, agonists or antagonists of thepresent invention. Also, polynucleotides or polypeptides, as well asagonists or antagonists of the present invention, could be used tostimulate the proliferation of and differentiation of type IIpneumocytes, which may help treat or prevent disease such as hyalinemembrane diseases, such as infant respiratory distress syndrome andbronchopulmonary displasia, in premature infants.

[0623] Polynucleotides or polypeptides, as well as agonists orantagonists of the present invention, could stimulate the proliferationand differentiation of hepatocytes and, thus, could be used to alleviateor treat liver diseases and pathologies such as fulminant liver failurecaused by cirrhosis, liver damage caused by viral hepatitis and toxicsubstances (i.e., acetaminophen, carbon tetraholoride and otherhepatotoxins known in the art).

[0624] In addition, polynucleotides or polypeptides, as well as agonistsor antagonists of the present invention, could be used treat or preventthe onset of diabetes mellitus. In patients with newly diagnosed Types Iand II diabetes, where some islet cell function remains, polynucleotidesor polypeptides, as well as agonists or antagonists of the presentinvention, could be used to maintain the islet function so as toalleviate, delay or prevent permanent manifestation of the disease.Also, polynucleotides or polypeptides, as well as agonists orantagonists of the present invention, could be used as an auxiliary inislet cell transplantation to improve or promote islet cell function.

[0625] Neural Activity and Neurological Diseases

[0626] The polynucleotides, polypeptides and agonists or antagonists ofthe invention may be used for the diagnosis and/or treatment ofdiseases, disorders, damage or injury of the brain and/or nervoussystem. Nervous system disorders that can be treated with thecompositions of the invention (e.g., immunoglobulin superfamilypolypeptides, polynucleotides, and/or agonists or antagonists), include,but are not limited to, nervous system injuries, and diseases ordisorders which result in either a disconnection of axons, a diminutionor degeneration of neurons, or demyelination. Nervous system lesionswhich may be treated in a patient (including human and non-humanmammalian patients) according to the methods of the invention, includebut are not limited to, the following lesions of either the central(including spinal cord, brain) or peripheral nervous systems: (1)ischemic lesions, in which a lack of oxygen in a portion of the nervoussystem results in neuronal injury or death, including cerebralinfarction or ischemia, or spinal cord infarction or ischemia; (2)traumatic lesions, including lesions caused by physical injury orassociated with surgery, for example, lesions which sever a portion ofthe nervous system, or compression injuries; (3) malignant lesions, inwhich a portion of the nervous system is destroyed or injured bymalignant tissue which is either a nervous system associated malignancyor a malignancy derived from non-nervous system tissue; (4) infectiouslesions, in which a portion of the nervous system is destroyed orinjured as a result of infection, for example, by an abscess orassociated with infection by human immunodeficiency virus, herpeszoster, or herpes simplex virus or with Lyme disease, tuberculosis, orsyphilis; (5) degenerative lesions, in which a portion of the nervoussystem is destroyed or injured as a result of a degenerative processincluding but not limited to, degeneration associated with Parkinson'sdisease, Alzheimer's disease, Huntington's chorea, or amyotrophiclateral sclerosis (ALS); (6) lesions associated with nutritionaldiseases or disorders, in which a portion of the nervous system isdestroyed or injured by a nutritional disorder or disorder of metabolismincluding, but not limited to, vitamin B12 deficiency, folic aciddeficiency, Wernicke disease, tobacco-alcohol amblyopia,Marchiafava-Bignami disease (primary degeneration of the corpuscallosum), and alcoholic cerebellar degeneration; (7) neurologicallesions associated with systemic diseases including, but not limited to,diabetes (diabetic neuropathy, Bell's palsy), systemic lupuserythematosus, carcinoma, or sarcoidosis; (8) lesions caused by toxicsubstances including alcohol, lead, or particular neurotoxins; and (9)demyelinated lesions in which a portion of the nervous system isdestroyed or injured by a demyelinating disease including, but notlimited to, multiple sclerosis, human immunodeficiency virus-associatedmyelopathy, transverse myelopathy or various etiologies, progressivemultifocal leukoencephalopathy, and central pontine myelinolysis.

[0627] In one embodiment, the polypeptides, polynucleotides, or agonistsor antagonists of the invention are used to protect neural cells fromthe damaging effects of hypoxia. In a further preferred embodiment, thepolypeptides, polynucleotides, or agonists or antagonists of theinvention are used to protect neural cells from the damaging effects ofcerebral hypoxia. According to this embodiment, the compositions of theinvention are used to treat or prevent neural cell injury associatedwith cerebral hypoxia. In one non-exclusive aspect of this embodiment,the polypeptides, polynucleotides, or agonists or antagonists of theinvention, are used to treat or prevent neural cell injury associatedwith cerebral ischemia. In another non-exclusive aspect of thisembodiment, the polypeptides, polynucleotides, or agonists orantagonists of the invention are used to treat or prevent neural cellinjury associated with cerebral infarction.

[0628] In another preferred embodiment, the polypeptides,polynucleotides, or agonists or antagonists of the invention are used totreat or prevent neural cell injury associated with a stroke. In aspecific embodiment, the polypeptides, polynucleotides, or agonists orantagonists of the invention are used to treat or prevent cerebralneural cell injury associated with a stroke.

[0629] In another preferred embodiment, the polypeptides,polynucleotides, or agonists or antagonists of the invention are used totreat or prevent neural cell injury associated with a heart attack. In aspecific embodiment, the polypeptides, polynucleotides, or agonists orantagonists of the invention are used to treat or prevent cerebralneural cell injury associated with a heart attack.

[0630] The compositions of the invention which are useful for treatingor preventing a nervous system disorder may be selected by testing forbiological activity in promoting the survival or differentiation ofneurons. For example, and not by way of limitation, compositions of theinvention which elicit any of the following effects may be usefulaccording to the invention: (1) increased survival time of neurons inculture either in the presence or absence of hypoxia or hypoxicconditions; (2) increased sprouting of neurons in culture or in vivo;(3) increased production of a neuron-associated molecule in culture orin vivo, e.g., choline acetyltransferase or acetylcholinesterase withrespect to motor neurons; or (4) decreased symptoms of neurondysfunction in vivo. Such effects may be measured by any method known inthe art. In preferred, non-limiting embodiments, increased survival ofneurons may routinely be measured using a method set forth herein orotherwise known in the art, such as, for example, in Zhang et al., ProcNatl Acad Sci USA 97:3637-42 (2000) or in Arakawa et al., J. Neurosci.,10:3507-15 (1990); increased sprouting of neurons may be detected bymethods known in the art, such as, for example, the methods set forth inPestronk et al., Exp. Neurol., 70:65-82 (1980), or Brown et al., Ann.Rev. Neurosci., 4:17-42 (1981); increased production ofneuron-associated molecules may be measured by bioassay, enzymaticassay, antibody binding, Northern blot assay, etc., using techniquesknown in the art and depending on the molecule to be measured; and motorneuron dysfunction may be measured by assessing the physicalmanifestation of motor neuron disorder, e.g., weakness, motor neuronconduction velocity, or functional disability.

[0631] In specific embodiments, motor neuron disorders that may betreated according to the invention include, but are not limited to,disorders such as infarction, infection, exposure to toxin, trauma,surgical damage, degenerative disease or malignancy that may affectmotor neurons as well as other components of the nervous system, as wellas disorders that selectively affect neurons such as amyotrophic lateralsclerosis, and including, but not limited to, progressive spinalmuscular atrophy, progressive bulbar palsy, primary lateral sclerosis,infantile and juvenile muscular atrophy, progressive bulbar paralysis ofchildhood (Fazio-Londe syndrome), poliomyelitis and the post poliosyndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-ToothDisease).

[0632] Further, polypeptides or polynucleotides of the invention mayplay a role in neuronal survival; synapse formation; conductance; neuraldifferentiation, etc. Thus, compositions of the invention (includingimmunoglobulin superfamily polynucleotides, polypeptides, and agonistsor antagonists) may be used to diagnose and/or treat or prevent diseasesor disorders associated with these roles, including, but not limited to,learning and/or cognition disorders. The compositions of the inventionmay also be useful in the treatment or prevention of neurodegenerativedisease states and/or behavioural disorders. Such neurodegenerativedisease states and/or behavioral disorders include, but are not limitedto, Alzheimers Disease, Parkinsons Disease, Huntingtons Disease,Tourette Syndrome, schizophrenia, mania, dementia, paranoia, obsessivecompulsive disorder, panic disorder, learning disabilities, ALS,psychoses, autism, and altered behaviors, including disorders infeeding, sleep patterns, balance, and perception. In addition,compositions of the invention may also play a role in the treatment,prevention and/or detection of developmental disorders associated withthe developing embryo, or sexually-linked disorders.

[0633] Additionally, polypeptides, polynucleotides and/or agonists orantagonists of the invention, may be useful in protecting neural cellsfrom diseases, damage, disorders, or injury, associated withcerebrovascular disorders including, but not limited to, carotid arterydiseases (e.g., carotid artery thrombosis, carotid stenosis, or MoyamoyaDisease), cerebral amyloid angiopathy, cerebral aneurysm, cerebralanoxia, cerebral arteriosclerosis, cerebral arteriovenous malformations,cerebral artery diseases, cerebral embolism and thrombosis (e.g.,carotid artery thrombosis, sinus thrombosis, or Wallenberg's Syndrome),cerebral hemorrhage (e.g., epidural or subdural hematoma, orsubarachnoid hemorrhage), cerebral infarction, cerebral ischemia (e.g.,transient cerebral ischemia, Subclavian Steal Syndrome, orvertebrobasilar insufficiency), vascular dementia (e.g., multi-infarct),leukomalacia, periventricular, and vascular headache (e.g., clusterheadache or migraines).

[0634] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing polynucleotides orpolypeptides, as well as agonists or antagonists of the presentinvention, for therapeutic purposes, for example, to stimulateneurological cell proliferation and/or differentiation. Therefore,polynucleotides, polypeptides, agonists and/or antagonists of theinvention may be used to treat and/or detect neurologic diseases.Moreover, polynucleotides or polypeptides, or agonists or antagonists ofthe invention, can be used as a marker or detector of a particularnervous system disease or disorder.

[0635] Examples of neurologic diseases which can be treated or detectedwith polynucleotides, polypeptides, agonists, and/or antagonists of thepresent invention include brain diseases, such as metabolic braindiseases which includes phenylketonuria such as maternalphenylketonuria, pyruvate carboxylase deficiency, pyruvate dehydrogenasecomplex deficiency, Wernicke's Encephalopathy, brain edema, brainneoplasms such as cerebellar neoplasms which include infratentorialneoplasms, cerebral ventricle neoplasms such as choroid plexusneoplasms, hypothalamic neoplasms, supratentorial neoplasms, canavandisease, cerebellar diseases such as cerebellar ataxia which includespinocerebellar degeneration such as ataxia telangiectasia, cerebellardyssynergia, Friederich's Ataxia, Machado-Joseph Disease,olivopontocerebellar atrophy, cerebellar neoplasms such asinfratentorial neoplasms, diffuse cerebral sclerosis such asencephalitis periaxialis, globoid cell leukodystrophy, metachromaticleukodystrophy and subacute sclerosing panencephalitis.

[0636] Additional neurologic diseases which can be treated or detectedwith polynucleotides, polypeptides, agonists, and/or antagonists of thepresent invention include cerebrovascular disorders (such as carotidartery diseases which include carotid artery thrombosis, carotidstenosis arid Moyanioya Disease), cerebral amyloid angiopathy, cerebralaneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebralarteriovenous malformations, cerebral artery diseases, cerebral embolismand thrombosis such as carotid artery thrombosis, sinus thrombosis andWallenberg's Syndrome, cerebral hemorrhage such as epidural hematoma,subdural hematoma and subarachnoid hemorrhage, cerebral infarction,cerebral ischemia such as transient cerebral ischemia, Subclavian StealSyndrome and vertebrobasilar insufficiency, vascular dementia such asmulti-infarct dementia, periventricular leukomalacia, vascular headachesuch as cluster headache and migraine.

[0637] Additional neurologic diseases which can be treated or detectedwith polynucleotides, polypeptides, agonists, and/or antagonists of thepresent invention include dementia such as AIDS Dementia Complex,presenile dementia such as Alzheimer's Disease and Creutzfeldt-JakobSyndrome, senile dementia such as Alzheimer's Disease and progressivesupranuclear palsy, vascular dementia such as multi-infarct dementia,encephalitis which include encephalitis periaxialis, viral encephalitissuch as epidemic encephalitis, Japanese Encephalitis, St. LouisEncephalitis, tick-borne encephalitis and West Nile Fever, acutedisseminated encephalomyelitis, meningoencephalitis such asuveomeningoencephalitic syndrome, Postencephalitic Parkinson Disease andsubacute sclerosing panencephalitis, encephalomalacia such asperiventricular leukomalacia, epilepsy such as generalized epilepsywhich includes infantile spasms, absence epilepsy, myoclonic epilepsywhich includes MERRF Syndrome, tonic-clonic epilepsy, partial epilepsysuch as complex partial epilepsy, frontal lobe epilepsy and temporallobe epilepsy, post-traumatic epilepsy, status epilepticus such asEpilepsia Partialis Continua, and Hallervorden-Spatz Syndrome.

[0638] Additional neurologic diseases which can be treated or detectedwith polynucleotides, polypeptides, agonists, and/or antagonists of thepresent invention include hydrocephalus such as Dandy-Walker Syndromeand normal pressure hydrocephalus, hypothalamic diseases such ashypothalamic neoplasms, cerebral malaria, narcolepsy which includescataplexy, bulbar poliomyelitis, cerebri pseudotumor, Rett Syndrome,Reye's Syndrome, thalamic diseases, cerebral toxoplasmosis, intracranialtuberculoma and Zellweger Syndrome, central nervous system infectionssuch as AIDS Dementia Complex, Brain Abscess, subdural empyema,encephalomyelitis such as Equine Encephalomyelitis, Venezuelan EquineEncephalomyelitis, Necrotizing Hemorrhagic Encephalomyelitis, Visna, andcerebral malaria.

[0639] Additional neurologic diseases which can be treated or detectedwith polynucleotides, polypeptides, agonists, and/or antagonists of thepresent invention include meningitis such as arachnoiditis, asepticmeningtitis such as viral meningtitis which includes lymphocyticchoriomeningitis, Bacterial meningtitis which includes HaemophilusMeningtitis, Listeria Meningtitis, Meningococcal Meningtitis such asWaterhouse-Friderichsen Syndrome, Pneumococcal Meningtitis and meningealtuberculosis, fungal meningitis such as Cryptococcal Meningtitis,subdural effusion, meningoencephalitis such as uvemeningoencephaliticsyndrome, myelitis such as transverse myelitis, neurosyphilis such astabes dorsalis, poliomyelitis which includes bulbar poliomyelitis andpostpoliomyelitis syndrome, prion diseases (such as Creutzfeldt-JakobSyndrome, Bovine Spongiform Encephalopathy, Gerstmann-StrausslerSyndrome, Kuru, Scrapie), and cerebral toxoplasmosis.

[0640] Additional neurologic diseases which can be treated or detectedwith polynucleotides, polypeptides, agonists, and/or antagonists of thepresent invention include central nervous system neoplasms such as brainneoplasms that include cerebellar neoplasms such as infratentorialneoplasms, cerebral ventricle neoplasms such as choroid plexusneoplasms, hypothalamic neoplasms and supratentorial neoplasms,meningeal neoplasms, spinal cord neoplasms which include epiduralneoplasms, demyelinating diseases such as Canavan Diseases, diffusecerebral sceloris which includes adrenoleukodystrophy, encephalitisperiaxialis, globoid cell leukodystrophy, diffuse cerebral sclerosissuch as metachromatic leukodystrophy, allergic encephalomyelitis,necrotizing hemorrhagic encephalomyelitis, progressive multifocalleukoencephalopathy, multiple sclerosis, central pontine myelinolysis,transverse myelitis, neuromyelitis optica, Scrapie, Swayback, ChronicFatigue Syndrome, Visna, High Pressure Nervous Syndrome, Meningism,spinal cord diseases such as amyotonia congenita, amyotrophic lateralsclerosis, spinal muscular atrophy such as Werdnig-Hoffmann Disease,spinal cord compression, spinal cord neoplasms such as epiduralneoplasms, syringomyelia, Tabes Dorsalis, Stiff-Man Syndrome, mentalretardation such as Angelman Syndrome, Cri-du-Chat Syndrome, De Lange'sSyndrome, Down Syndrome, Gangliosidoses such as gangliosidoses G(Ml),Sandhoff Disease, Tay-Sachs Disease, Hartnup Disease, homocystinuria,Laurence-Moon-Biedl Syndrome, Lesch-Nyhan Syndrome, Maple Syrup UrineDisease, mucolipidosis such as fucosidosis, neuronalceroid-lipofuscinosis, oculocerebrorenal syndrome, phenylketonuria suchas maternal phenylketonuria, Prader-Willi Syndrome, Rett Syndrome,Rubinstein-Taybi Syndrome, Tuberous Sclerosis, WAGR Syndrome, nervoussystem abnormalities such as holoprosencephaly, neural tube defects suchas anencephaly which includes hydrangencephaly, Arnold-Chairi Deformity,encephalocele, meningocele, meningomyelocele, spinal dysraphism such asspina bifida cystica and spina bifida occulta.

[0641] Additional neurologic diseases which can be treated or detectedwith polynucleotides, polypeptides, agonists, and/or antagonists of thepresent invention include hereditary motor and sensory neuropathieswhich include Charcot-Marie Disease, Hereditary optic atrophy, Refsum'sDisease, hereditary spastic paraplegia, Werdnig-Hoffmann Disease,Hereditary Sensory and Autonomic Neuropathies such as CongenitalAnalgesia and Familial Dysautonomia, Neurologic manifestations (such asagnosia that include Gerstmann's Syndrome, Amnesia such as retrogradeamnesia, apraxia, neurogenic bladder, cataplexy, communicative disorderssuch as hearing disorders that includes deafness, partial hearing loss,loudness recruitment and tinnitus, language disorders such as aphasiawhich include agraphia, anomia, broca aphasia, and Wernicke Aphasia,Dyslexia such as Acquired Dyslexia, language development disorders,speech disorders such as aphasia which includes anomia, broca aphasiaand Wernicke Aphasia, articulation disorders, communicative disorderssuch as speech disorders which include dysarthria, echolalia, mutism andstuttering, voice disorders such as aphonia and hoarseness, decerebratestate, delirium, fasciculation, hallucinations, meningism, movementdisorders such as angelman syndrome, ataxia, athetosis, chorea,dystonia, hypokinesia, muscle hypotonia, myoclonus, tic, torticollis andtremor, muscle hypertonia such as muscle rigidity such as stiff-mansyndrome, muscle spasticity, paralysis such as facial paralysis whichincludes Herpes Zoster Oticus, Gastroparesis, Hemiplegia,ophthalmoplegia such as diplopia, Duane's Syndrome, Homer's Syndrome,Chronic progressive external ophthalmoplegia such as Keams Syndrome,Bulbar Paralysis, Tropical Spastic Paraparesis, Paraplegia such asBrown-Sequard Syndrome, quadriplegia, respiratory paralysis and vocalcord paralysis, paresis, phantom limb, taste disorders such as ageusiaand dysgeusia, vision disorders such as amblyopia, blindness, colorvision defects, diplopia, hemianopsia, scotoma and subnormal vision,sleep disorders such as hypersomnia which includes Kleine-LevinSyndrome, insomnia, and somnambulism, spasm such as trismus,unconsciousness such as coma, persistent vegetative state and syncopeand vertigo, neuromuscular diseases such as amyotonia congenita,amyotrophic lateral sclerosis, Lambert-Eaton Myasthenic Syndrome, motorneuron disease, muscular atrophy such as spinal muscular atrophy,Charcot-Marie Disease and Werdnig-Hoffmann Disease, PostpoliomyelitisSyndrome, Muscular Dystrophy, Myasthenia Gravis, Myotonia Atrophica,Myotonia Confenita, Nemaline Myopathy, Familial Periodic Paralysis,Multiplex Paramyloclonus, Tropical Spastic Paraparesis and Stiff-ManSyndrome, peripheral nervous system diseases such as acrodynia, amyloidneuropathies, autonomic nervous system diseases such as Adie's Syndrome,Barre-Lieou Syndrome, Familial Dysautonomia, Horner's Syndrome, ReflexSympathetic Dystrophy and Shy-Drager Syndrome, Cranial Nerve Diseasessuch as Acoustic Nerve Diseases such as Acoustic Neuroma which includesNeurofibromatosis 2, Facial Nerve Diseases such as Facial Neuralgia,Melkersson-Rosenthal Syndrome, ocular motility disorders which includesamblyopia, nystagmus, oculomotor nerve paralysis, ophthalmoplegia suchas Duane's Syndrome, Horner's Syndrome, Chronic Progressive ExternalOphthalmoplegia which includes Keams Syndrome, Strabismus such asEsotropia and Exotropia, Oculomotor Nerve Paralysis, Optic NerveDiseases such as Optic Atrophy which includes Hereditary Optic Atrophy,Optic Disk Drusen, Optic Neuritis such as Neuromyelitis Optica,Papilledema, Trigeminal Neuralgia, Vocal Cord Paralysis, DemyelinatingDiseases such as Neuromyelitis Optica and Swayback, and Diabeticneuropathies such as diabetic foot.

[0642] Additional neurologic diseases which can be treated or detectedwith polynucleotides, polypeptides, agonists, and/or antagonists of thepresent invention include nerve compression syndromes such as carpaltunnel syndrome, tarsal tunnel syndrome, thoracic outlet syndrome suchas cervical rib syndrome, ulnar nerve compression syndrome, neuralgiasuch as causalgia, cervico-brachial neuralgia, facial neuralgia andtrigeminal neuralgia, neuritis such as experimental allergic neuritis,optic neuritis, polyneuritis, polyradiculoneuritis and radiculities suchas polyradiculitis, hereditary motor and sensory neuropathies such asCharcot-Marie Disease, Hereditary Optic Atrophy, Refsum's Disease,Hereditary Spastic Paraplegia and Werdnig-Hoffmann Disease, HereditarySensory and Autonomic Neuropathies which include Congenital Analgesiaand Familial Dysautonomia, POEMS Syndrome, Sciatica, Gustatory Sweatingand Tetany).

[0643] Infectious Disease

[0644] Polynucleotides or polypeptides, as well as agonists orantagonists of the present invention can be used to treat or detectinfectious agents. For example, by increasing the immune response,particularly increasing the proliferation and differentiation of Band/or T cells, infectious diseases may be treated. The immune responsemay be increased by either enhancing an existing immune response, or byinitiating a new immune response. Alternatively, polynucleotides orpolypeptides, as well as agonists or antagonists of the presentinvention may also directly inhibit the infectious agent, withoutnecessarily eliciting an immune response.

[0645] Viruses are one example of an infectious agent that can causedisease or symptoms that can be treated or detected by a polynucleotideor polypeptide and/or agonist or antagonist of the present invention.Examples of viruses, include, but are not limited to Examples ofviruses, include, but are not limited to the following DNA and RNAviruses and viral families: Arbovirus, Adenoviridae, Arenaviridae,Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae,Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae(Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex,Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus,Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A, Influenza B, andparainfluenza), Papiloma virus, Papovaviridae, Parvoviridae,Picornaviridae, Poxviridae (such as Smallpox or Vaccinia), Reoviridae(e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), andTogaviridae (e.g., Rubivirus). Viruses falling within these families cancause a variety of diseases or symptoms, including, but not limited to:arthritis, bronchiollitis, respiratory syncytial virus, encephalitis,eye infections (e.g., conjunctivitis, keratitis), chronic fatiguesyndrome, hepatitis (A, B, C, E, Chronic Active, Delta), Japanese Bencephalitis, Junin, Chikungunya, Rift Valley fever, yellow fever,meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt'sLymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza,Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitteddiseases, skin diseases (e.g., Kaposi's, warts), and viremia.polynucleotides or polypeptides, or agonists or antagonists of theinvention, can be used to treat or detect any of these symptoms ordiseases. In specific embodiments, polynucleotides, polypeptides, oragonists or antagonists of the invention are used to treat: meningitis,Dengue, EBV, and/or hepatitis (e.g., hepatitis B). In an additionalspecific embodiment polynucleotides, polypeptides, or agonists orantagonists of the invention are used to treat patients nonresponsive toone or more other commercially available hepatitis vaccines. In afurther specific embodiment polynucleotides, polypeptides, or agonistsor antagonists of the invention are used to treat AIDS.

[0646] Similarly, bacterial or fungal agents that can cause disease orsymptoms and that can be treated or detected by a polynucleotide orpolypeptide and/or agonist or antagonist of the present inventioninclude, but not limited to, include, but not limited to, the followingGram-Negative and Gram-positive bacteria and bacterial families andfungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium,Norcardia), Cryptococcus neoformans, Aspergillosis, Bacillaceae (e.g.,Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella,Borrelia (e.g., Borrelia burgdorferi, Brucellosis, Candidiasis,Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses, E.coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic E. coli),Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, andSalmonella paratyphi), Serratia, Yersinia), Erysipelothrix,Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales,Mycobacterium leprae, Vibrio cholerae, Neisseriaceae (e.g.,Acinetobacter, Gonorrhea, Menigococcal), Meisseria meningitidis,Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus (e.g.,Heamophilus influenza type B), Pasteurella), Pseudomonas,Rickettsiaceae, Chlamydiaceae, Syphilis, Shigella spp., Staphylococcal,Meningiococcal, Pneumococcal and Streptococcal (e.g., Streptococcuspneumoniae and Group B Streptococcus). These bacterial or fungalfamilies can cause the following diseases or symptoms, including, butnot limited to: bacteremia, endocarditis, eye infections(conjunctivitis, tuberculosis, uveitis), gingivitis, opportunisticinfections (e.g., AIDS related infections), paronychia,prosthesis-related infections, Reiter's Disease, respiratory tractinfections, such as Whooping Cough or Empyema, sepsis, Lyme Disease,Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning,Typhoid, pneumonia, Gonorrhea, meningitis (e.g., mengitis types A andB), Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, RheumaticFever, Scarlet Fever, sexually transmitted diseases, skin diseases(e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections,wound infections. Polynucleotides or polypeptides, agonists orantagonists of the invention, can be used to treat or detect any ofthese symptoms or diseases. In specific embodiments, Ppolynucleotides,potypeptides, agonists or antagonists of the invention are used totreat: tetanus, Diptheria, botulism, and/or meningitis type B.

[0647] Moreover, parasitic agents causing disease or symptoms that canbe treated or detected by a polynucleotide or polypeptide and/or agonistor antagonist of the present invention include, but not limited to, thefollowing families or class: Amebiasis, Babesiosis, Coccidiosis,Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis,Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis,Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax,Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale). Theseparasites can cause a variety of diseases or symptoms, including, butnot limited to: Scabies, Trombiculiasis, eye infections, intestinaldisease (e.g., dysentery, giardiasis), liver disease, lung disease,opportunistic infections (e.g., AIDS related), malaria, pregnancycomplications, and toxoplasmosis. polynucleotides or polypeptides, oragonists or antagonists of the invention, can be used to treat or detectany of these symptoms or diseases.

[0648] Polynucleotides or polypeptides, as well as agonists orantagonists of the present invention of the present invention couldeither be by administering an effective amount of a polypeptide to thepatient, or by removing cells from the patient, supplying the cells witha polynucleotide of the present invention, and returning the engineeredcells to the patient (ex vivo therapy). Moreover, the polypeptide orpolynucleotide of the present invention can be used as an antigen in avaccine to raise an immune response against infectious disease.

[0649] Regeneration

[0650] Polynucleotides or polypeptides, as well as agonists orantagonists of the present invention can be used to differentiate,proliferate, and attract cells, leading to the regeneration of tissues.(See, Science 276:59-87 (1997).) The regeneration of tissues could beused to repair, replace, or protect tissue damaged by congenitaldefects, trauma (wounds, burns, incisions, or ulcers), age, disease(e.g. osteoporosis, osteocarthritis, periodontal disease, liverfailure), surgery, including cosmetic plastic surgery, fibrosis,reperfusion injury, or systemic cytokine damage.

[0651] Tissues that could be regenerated using the present inventioninclude organs (e.g., pancreas, liver, intestine, kidney, skin,endothelium), muscle (smooth, skeletal or cardiac), vasculature(including vascular and lymphatics), nervous, hematopoietic, andskeletal (bone, cartilage, tendon, and ligament) tissue. Preferably,regeneration occurs without or decreased scarring. Regeneration also mayinclude angiogenesis.

[0652] Moreover, polynucleotides or polypeptides, as well as agonists orantagonists of the present invention, may increase regeneration oftissues difficult to heal. For example, increased tendon/ligamentregeneration would quicken recovery time after damage. Polynucleotidesor polypeptides, as well as agonists or antagonists of the presentinvention could also be used prophylactically in an effort to avoiddamage. Specific diseases that could be treated include of tendinitis,carpal tunnel syndrome, and other tendon or ligament defects. A furtherexample of tissue regeneration of non-healing wounds includes pressureulcers, ulcers associated with vascular insufficiency, surgical, andtraumatic wounds.

[0653] Similarly, nerve and brain tissue could also be regenerated byusing polynucleotides or polypeptides, as well as agonists orantagonists of the present invention, to proliferate and differentiatenerve cells. Diseases that could be treated using this method includecentral and peripheral nervous system diseases, neuropathies, ormechanical and traumatic disorders (e.g., spinal cord disorders, headtrauma, cerebrovascular disease, and stoke). Specifically, diseasesassociated with peripheral nerve injuries, peripheral neuropathy (e.g.,resulting from chemotherapy or other medical therapies), localizedneuropathies, and central nervous system diseases (e.g., Alzheimer'sdisease, Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, and Shy-Drager syndrome), could all be treated using thepolynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention.

[0654] Chemotaxis

[0655] Polynucleotides or polypeptides, as well as agonists orantagonists of the present invention may have chemotaxis activity. Achemotaxic molecule attracts or mobilizes cells (e.g., monocytes,fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelialand/or endothelial cells) to a particular site in the body, such asinflammation, infection, or site of hyperproliferation. The mobilizedcells can then fight off and/or heal the particular trauma orabnormality.

[0656] Polynucleotides or polypeptides, as well as agonists orantagonists of the present invention may increase chemotaxic activity ofparticular cells. These chemotactic molecules can then be used to treatinflammation, infection, hyperproliferative disorders, or any immunesystem disorder by increasing the number of cells targeted to aparticular location in the body. For example, chemotaxic molecules canbe used to treat wounds and other trauma to tissues by attracting immunecells to the injured location. Chemotactic molecules of the presentinvention can also attract fibroblasts, which can be used to treatwounds.

[0657] It is also contemplated that polynucleotides or polypeptides, aswell as agonists or antagonists of the present invention may inhibitchemotactic activity. These molecules could also be used to treatdisorders. Thus, polynucleotides or polypeptides, as well as agonists orantagonists of the present invention could be used as an inhibitor ofchemotaxis.

[0658] Binding Activity

[0659] A polypeptide of the present invention may be used to screen formolecules that bind to the polypeptide or for molecules to which thepolypeptide binds. The binding of the polypeptide and the molecule mayactivate (agonist), increase, inhibit (antagonist), or decrease activityof the polypeptide or the molecule bound. Examples of such moleculesinclude antibodies, oligonucleotides, proteins (e.g., receptors),orsmall molecules.

[0660] Preferably, the molecule is closely related to the natural ligandof the polypeptide, e.g., a fragment of the ligand, or a naturalsubstrate, a ligand, a structural or functional mimetic. (See, Coliganet al., Current Protocols in Immunology 1(2):Chapter 5 (1991).)Similarly, the molecule can be closely related to the natural receptorto which the polypeptide binds, or at least, a fragment of the receptorcapable of being bound by the polypeptide (e.g., active site). In eithercase, the molecule can be rationally designed using known techniques.

[0661] Preferably, the screening for these molecules involves producingappropriate cells which express the polypeptide. Preferred cells includecells from mammals, yeast, Drosophila, or E. coli. Cells expressing thepolypeptide (or cell membrane containing the expressed polypeptide) arethen preferably contacted with a test compound potentially containingthe molecule to observe binding, stimulation, or inhibition of activityof either the polypeptide or the molecule.

[0662] The assay may simply test binding of a candidate compound to thepolypeptide, wherein binding is detected by a label, or in an assayinvolving competition with a labeled competitor. Further, the assay maytest whether the candidate compound results in a signal generated bybinding to the polypeptide.

[0663] Alternatively, the assay can be carried out using cell-freepreparations, polypeptide/molecule affixed to a solid support, chemicallibraries, or natural product mixtures. The assay may also simplycomprise the steps of mixing a candidate compound with a solutioncontaining a polypeptide, measuring polypeptide/molecule activity orbinding, and comparing the polypeptide/molecule activity or binding to astandard.

[0664] Preferably, an ELISA assay can measure polypeptide level oractivity in a sample (e.g., biological sample) using a monoclonal orpolyclonal antibody. The antibody can measure polypeptide level oractivity by either binding, directly or indirectly, to the polypeptideor by competing with the polypeptide for a substrate.

[0665] Additionally, the receptor to which the polypeptide of thepresent invention binds can be identified by numerous methods known tothose of skill in the art, for example, ligand panning and FACS sorting(Coligan, et al., Current Protocols in Immun., 1(2), Chapter 5, (1991)).For example, expression cloning is employed wherein polyadenylated RNAis prepared from a cell responsive to the polypeptides, for example,NIH3T3 cells which are known to contain multiple receptors for the FGFfamily proteins, and SC-3 cells, and a cDNA library created from thisRNA is divided into pools and used to transfect COS cells or other cellsthat are not responsive to the polypeptides. Transfected cells which aregrown on glass slides are exposed to the polypeptide of the presentinvention, after they have been labelled. The polypeptides can belabeled by a variety of means including iodination or inclusion of arecognition site for a site-specific protein kinase.

[0666] Following fixation and incubation, the slides are subjected toauto-radiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an iterative sub-pooling andre-screening process, eventually yielding a single clones that encodesthe putative receptor.

[0667] As an alternative approach for receptor identification, thelabeled polypeptides can be photoaffinity linked with cell membrane orextract preparations that express the receptor molecule. Cross-linkedmaterial is resolved by PAGE analysis and exposed to X-ray film. Thelabeled complex containing the receptors of the polypeptides can beexcised, resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide S probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

[0668] Moreover, the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”) may be employed to modulate the activities of thepolypeptide of the present invention thereby effectively generatingagonists and antagonists of the polypeptide of the present invention.See generally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721,5,834,252, and 5,837,458, and Patten, P. A., et al, Curr. OpinionBiotechnol. 8:724-33 (1997); Harayama, S. Trends Biotechnol. 16(2):76-82(1998); Hansson, L. O., et al., J. Mol. Biol. 287:265-76 (1999); andLorenzo, M. M. and Blasco, R. Biotechniques 24(2):308-13 (1998) (each ofthese patents and publications are hereby incorporated by reference). Inone embodiment, alteration of polynucleotides and correspondingpolypeptides may be achieved by DNA shuffling. DNA shuffling involvesthe assembly of two or more DNA segments into a desired molecule byhomologous, or site-specific, recombination. In another embodiment,polynucleotides and corresponding polypeptides may be alterred by beingsubjected to random mutagenesis by error-prone PCR, random nucleotideinsertion or other methods prior to recombination. In anotherembodiment, one or more components, motifs, sections, parts, domains,fragments, etc., of the polypeptide of the present invention may berecombined with one or more components, motifs, sections, parts,domains, fragments, etc. of one or more heterologous molecules. Inpreferred embodiments, the heterologous molecules are family members. Infurther preferred embodiments, the heterologous molecule is a growthfactor such as, for example, platelet-derived growth factor (PDGF),insulin-like growth factor (IGF-I), transforming growth factor(TGF)-alpha, epidermal growth factor (EGF), fibroblast growth factor(FGF), TGF-beta, bone morphogenetic protein (BMP)-2, BMP-4, BMP-5,BMP-6, BMP-7, activins A and B, decapentaplegic(dpp), 60A, OP-2,dorsalin, growth differentiation factors (GDFs), nodal, MIS,inhibin-alpha, TGF-betal, TGF-beta2, TGF-beta3, TGF-beta5, andglial-derived neurotrophic factor (GDNF).

[0669] Other preferred fragments are biologically active fragments ofthe polypeptide of the present invention. Biologically active fragmentsare those exhibiting activity similar, but not necessarily identical, toan activity of the polypeptide of the present invention. The biologicalactivity of the fragments may include an improved desired activity, or adecreased undesirable activity.

[0670] Additionally, this invention provides a method of screeningcompounds to identify those which modulate the action of the polypeptideof the present invention. An example of such an assay comprisescombining a mammalian fibroblast cell, a the polypeptide of the presentinvention, the compound to be screened and ³[H] thymidine under cellculture conditions where the fibroblast cell would normally proliferate.A control assay may be performed in the absence of the compound to bescreened and compared to the amount of fibroblast proliferation in thepresence of the compound to determine if the compound stimulatesproliferation by determining the uptake of ³[H] thymidine in each case.The amount of fibroblast cell proliferation is measured by liquidscintillation chromatography which measures the incorporation of ³[H]thymidine. Both agonist and antagonist compounds may be identified bythis procedure.

[0671] In another method, a mammalian cell or membrane preparationexpressing a receptor for a polypeptide of the present invention isincubated with a labeled polypeptide of the present invention in thepresence of the compound. The ability of the compound to enhance orblock this interaction could then be measured. Alternatively, theresponse of a known second messenger system following interaction of acompound to be screened and the receptor is measured and the ability ofthe compound to bind to the receptor and elicit a second messengerresponse is measured to determine if the compound is a potential agonistor antagonist. Such second messenger systems include but are not limitedto, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.

[0672] All of these above assays can be used as diagnostic or prognosticmarkers. The molecules discovered using these assays can be used totreat disease or to bring about a particular result in a patient (e.g.,blood vessel growth) by activating or inhibiting thepolypeptide/molecule. Moreover, the assays can discover agents which mayinhibit or enhance the production of the polypeptides of the inventionfrom suitably manipulated cells or tissues.

[0673] Therefore, the invention includes a method of identifyingcompounds which bind to a polypeptide of the invention comprising thesteps of: (a) incubating a candidate binding compound with a polypeptideof the present invention; and (b) determining if binding has occurred.Moreover, the invention includes a method of identifyingagonists/antagonists comprising the steps of: (a) incubating a candidatecompound with a polypeptide of the present invention, (b) assaying abiological activity, and (b) determining if a biological activity of thepolypeptide has been altered.

[0674] Targeted Delivery

[0675] In another embodiment, the invention provides a method ofdelivering compositions to targeted cells expressing a receptor for apolypeptide of the invention, or cells expressing a cell bound form of apolypeptide of the invention.

[0676] As discussed herein, polypeptides or antibodies of the inventionmay be associated with heterologous polypeptides, heterologous nucleicacids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/orcovalent interactions. In one embodiment, the invention provides amethod for the specific delivery of compositions of the invention tocells by administering polypeptides of the invention (includingantibodies) that are associated with heterologous polypeptides ornucleic acids. In one example, the invention provides a method fordelivering a therapeutic protein into the targeted cell. In anotherexample, the invention provides a method for delivering a singlestranded nucleic acid (e.g., antisense or ribozymes) or double strandednucleic acid (e.g., DNA that can integrate into the cell's genome orreplicate episomally and that can be transcribed) into the targetedcell.

[0677] In another embodiment, the invention provides a method for thespecific destruction of cells (e.g., the destruction of tumor cells) byadministering polypeptides of the invention (e.g., polypeptides of theinvention or antibodies of the invention) in association with toxins orcytotoxic prodrugs.

[0678] By “toxin” is meant compounds that bind and activate endogenouscytotoxic effector systems, radioisotopes, holotoxins, modified toxins,catalytic subunits of toxins, or any molecules or enzymes not normallypresent in or on the surface of a cell that under defined conditionscause the cell's death. Toxins that may be used according to the methodsof the invention include, but are not limited to, radioisotopes known inthe art, compounds such as, for example, antibodies (or complementfixing containing portions thereof) that bind an inherent or inducedendogenous cytotoxic effector system, thymidine kinase, endonuclease,RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheriatoxin, saporin, momordin, gelonin, pokeweed antiviral protein,alpha-sarcin and cholera toxin. By “cytotoxic prodrug” is meant anon-toxic compound that is converted by an enzyme, normally present inthe cell, into a cytotoxic compound. Cytotoxic prodrugs that may be usedaccording to the methods of the invention include, but are not limitedto, glutamyl derivatives of benzoic acid mustard alkylating agent,phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside,daunorubisin, and phenoxyacetamide derivatives of doxorubicin.

[0679] Drug Screening

[0680] Further contemplated is the use of the polypeptides of thepresent invention, or the polynucleotides encoding these polypeptides,to screen for molecules which modify the activities of the polypeptidesof the present invention. Such a method would include contacting thepolypeptide of the present invention with a selected compound(s)suspected of having antagonist or agonist activity, and assaying theactivity of these polypeptides following binding.

[0681] This invention is particularly useful for screening therapeuticcompounds by using the polypeptides of the present invention, or bindingfragments thereof, in any of a variety of drug screening techniques. Thepolypeptide or fragment employed in such a test may be affixed to asolid support, expressed on a cell surface, free in solution, or locatedintracellularly. One method of drug screening utilizes eukaryotic orprokaryotic host cells which are stably transformed with recombinantnucleic acids expressing the polypeptide or fragment. Drugs are screenedagainst such transformed cells in competitive binding assays. One maymeasure, for example, the formulation of complexes between the agentbeing tested and a polypeptide of the present invention.

[0682] Thus, the present invention provides methods of screening fordrugs or any other agents which affect activities mediated by thepolypeptides of the present invention. These methods comprise contactingsuch an agent with a polypeptide of the present invention or a fragmentthereof and assaying for the presence of a complex between the agent andthe polypeptide or a fragment thereof, by methods well known in the art.In such a competitive binding assay, the agents to screen are typicallylabeled. Following incubation, free agent is separated from that presentin bound form, and the amount of free or uncomplexed label is a measureof the ability of a particular agent to bind to the polypeptides of thepresent invention.

[0683] Another technique for drug screening provides high throughputscreening for compounds having suitable binding affinity to thepolypeptides of the present invention, and is described in great detailin European Patent Application 84/03564, published on Sep. 13, 1984,which is incorporated herein by reference herein. Briefly stated, largenumbers of different small peptide test compounds are synthesized on asolid substrate, such as plastic pins or some other surface. The peptidetest compounds are reacted with polypeptides of the present inventionand washed. Bound polypeptides are then detected by methods well knownin the art. Purified polypeptides are coated directly onto plates foruse in the aforementioned drug screening techniques. In addition,non-neutralizing antibodies may be used to capture the peptide andimmobilize it on the solid support.

[0684] This invention also contemplates the use of competitive drugscreening assays in which neutralizing antibodies capable of bindingpolypeptides of the present invention specifically compete with a testcompound for binding to the polypeptides or fragments thereof. In thismanner, the antibodies are used to detect the presence of any peptidewhich shares one or more antigenic epitopes with a polypeptide of theinvention.

[0685] Antisense And Ribozyme (Antagonists)

[0686] In specific embodiments, antagonists according to the presentinvention are nucleic acids corresponding to the sequences contained inSEQ ID NO:X, or the complementary strand thereof, and/or to nucleotidesequences contained in the cDNA plasmid:Z identified in Table 1. In oneembodiment, antisense sequence is generated internally, by the organism,in another embodiment, the antisense sequence is separately administered(see, for example, O'Connor, J., Neurochem. 56:560 (1991).Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988). Antisense technology can be used tocontrol gene expression through antisense DNA or RNA, or throughtriple-helix formation. Antisense techniques are discussed for example,in Okano, J., Neurochem. 56:560 (1991); Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988). Triple helix formation is discussed in, for instance, Lee etal., Nucleic Acids Research 6:3073 (1979); Cooney et al., Science241:456 (1988); and Dervan et al., Science 251:1300 (1991). The methodsare based on binding of a polynucleotide to a complementary DNA or RNA.

[0687] For example, the use of c-myc and c-myb antisense RNA constructsto inhibit the growth of the non-lymphocytic leukemia cell line HL-60and other cell lines was previously described. (Wickstrom et al. (1988);Anfossi et al. (1989)). These experiments were performed in vitro byincubating cells with the oligoribonucleotide. A similar procedure forin vivo use is described in WO 91/15580. Briefly, a pair ofoligonucleotides for a given antisense RNA is produced as follows: Asequence complimentary to the first 15 bases of the open reading frameis flanked by an EcoR1 site on the 5 end and a HindIII site on the 3end. Next, the pair of oligonucleotides is heated at 90° C. for oneminute and then annealed in 2×ligation buffer (20 mM TRIS HCl pH 7.5, 10mM MgCl2, 10 MM dithiothreitol (DTT) and 0.2 mM ATP) and then ligated tothe EcoR1/Hind III site of the retroviral vector PMV7 (WO 91/15580).

[0688] For example, the 5′ coding portion of a polynucleotide thatencodes the polypeptide of the present invention may be used to designan antisense RNA oligonucleotide of from about 10 to 40 base pairs inlength. A DNA oligonucleotide is designed to be complementary to aregion of the gene involved in transcription thereby preventingtranscription and the production of the receptor. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into receptor polypeptide.

[0689] In one embodiment, the antisense nucleic acid of the invention isproduced intracellularly by transcription from an exogenous sequence.For example, a vector or a portion thereof, is transcribed, producing anantisense nucleic acid (RNA) of the invention. Such a vector wouldcontain a sequence encoding the antisense nucleic acid. Such a vectorcan remain episomal or become chromosomally integrated, as long as itcan be transcribed to produce the desired antisense RNA. Such vectorscan be constructed by recombinant DNA technology methods standard in theart. Vectors can be plasmid, viral, or others known in the art, used forreplication and expression in vertebrate cells. Expression of thesequence encoding the polypeptide of the present invnetion or fragmentsthereof, can be by any promoter known in the art to act in vertebrate,preferably human cells. Such promoters can be inducible or constitutive.Such promoters include, but are not limited to, the SV40 early promoterregion (Bernoist and Chambon, Nature 29:304-310 (1981), the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamotoet al., Cell 22:787-797 (1980), the herpes thymidine promoter (Wagner etal., Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445 (1981), the regulatorysequences of the metallothionein gene (Brinster, et al., Nature296:39-42 (1982)), etc.

[0690] The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a gene ofthe present invention. However, absolute complementarity, althoughpreferred, is not required. A sequence “complementary to at least aportion of an RNA,” referred to herein, means a sequence havingsufficient complementarity to be able to hybridize with the RNA, forminga stable duplex; in the case of double stranded antisense nucleic acids,a single strand of the duplex DNA may thus be tested, or triplexformation may be assayed. The ability to hybridize will depend on boththe degree of complementarity and the length of the antisense nucleicacid. Generally, the larger the hybridizing nucleic acid, the more basemismatches with a RNA it may contain and still form a stable duplex (ortriplex as the case may be). One skilled in the art can ascertain atolerable degree of mismatch by use of standard procedures to determinethe melting point of the hybridized complex.

[0691] Oligonucleotides that are complementary to the 5′ end of themessage, e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., 1994, Nature372:333-335. Thus, oligonucleotides complementary to either the 5′- or3′-non-translated, non-coding regions of polynucleotide sequencesdescribed herein could be used in an antisense approach to inhibittranslation of endogenous mRNA. Oligonucleotides complementary to the 5′untranslated region of the mRNA should include the complement of the AUGstart codon. Antisense oligonucleotides complementary to mRNA codingregions are less efficient inhibitors of translation but could be usedin accordance with the invention. Whether designed to hybridize to the5′-, 3′- or coding region of mRNA of the present invention, antisensenucleic acids should be at least six nucleotides in length, and arepreferably oligonucleotides ranging from 6 to about 50 nucleotides inlength. In specific aspects the oligonucleotide is at least 10nucleotides, at least 17 nucleotides, at least 25 nucleotides or atleast 50 nucleotides.

[0692] The polynucleotides of the invention can be DNA or RNA orchimeric mixtures or derivatives or modified versions thereof,single-stranded or double-stranded. The oligonucleotide can be modifiedat the base moiety, sugar moiety, or phosphate backbone, for example, toimprove stability of the molecule, hybridization, etc. Theoligonucleotide may include other appended groups such as peptides(e.g., for targeting host cell receptors in vivo), or agentsfacilitating transport across the cell membrane (see, e.g., Letsinger etal., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al.,1987, Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No. WO88/09810,published Dec. 15, 1988) or the blood-brain barrier (see, e.g., PCTPublication No. WO89/10134, published Apr. 25, 1988),hybridization-triggered cleavage agents. (See, e.g., Krol et al., 1988,BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon, 1988,Pharm. Res. 5:539-549). To this end, the oligonucleotide may beconjugated to another molecule, e.g., a peptide, hybridization triggeredcross-linking agent, transport agent, hybridization-triggered cleavageagent, etc.

[0693] The antisense oligonucleotide may comprise at least one modifiedbase moiety which is selected from the group including, but not limitedto, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

[0694] The antisense oligonucleotide may also comprise at least onemodified sugar moiety selected from the group including, but not limitedto, arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0695] In yet another embodiment, the antisense oligonucleotidecomprises at least one modified phosphate backbone selected from thegroup including, but not limited to, a phosphorothioate, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal or analog thereof.

[0696] In yet another embodiment, the antisense oligonucleotide is ana-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual b-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

[0697] Polynucleotides of the invention may be synthesized by standardmethods known in the art, e.g. by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligonucleotides may be synthesizedby the method of Stein et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

[0698] While antisense nucleotides complementary to the coding regionsequence could be used, those complementary to the transcribeduntranslated region are most preferred.

[0699] Potential antagonists according to the invention also includecatalytic RNA, or a ribozyme (See, e.g., PCT International PublicationWO 90/11364, published Oct. 4, 1990; Sarver et al, Science 247:1222-1225(1990). While ribozymes that cleave mRNA at site specific recognitionsequences can be used to destroy mRNAs, the use of hammerhead ribozymesis preferred. Hammerhead ribozymes cleave mRNAs at locations dictated byflanking regions that form complementary base pairs with the targetmRNA. The sole requirement is that the target mRNA have the followingsequence of two bases: 5′-UG-3′. The construction and production ofhammerhead ribozymes is well known in the art and is described morefully in Haseloff and Gerlach, Nature 334:585-591 (1988). There arenumerous potential hammerhead ribozyme cleavage sites within thenucleotide sequence of SEQ ID NO:X. Preferably, the ribozyme isengineered so that the cleavage recognition site is located near the 5′end of the mRNA; i.e., to increase efficiency and minimize theintracellular accumulation of non-functional mRNA transcripts.

[0700] As in the antisense approach, the ribozymes of the invention canbe composed of modified oligonucleotides (e.g. for improved stability,targeting, etc.) and should be delivered to cells which expresspolypeptides of the present invention in vivo. DNA constructs encodingthe ribozyme may be introduced into the cell in the same manner asdescribed above for the introduction of antisense encoding DNA. Apreferred method of delivery involves using a DNA construct “encoding”the ribozyme under the control of a strong constitutive promoter, suchas, for example, pol III or pol II promoter, so that transfected cellswill produce sufficient quantities of the ribozyme to destroy endogenousmessages and inhibit translation. Since ribozymes unlike antisensemolecules, are catalytic, a lower intracellular concentration isrequired for efficiency.

[0701] Antagonist/agonist compounds may be employed to inhibit the cellgrowth and proliferation effects of the polypeptides of the presentinvention on neoplastic cells and tissues, i.e. stimulation ofangiogenesis of tumors, and, therefore, retard or prevent abnormalcellular growth and proliferation, for example, in tumor formation orgrowth.

[0702] The antagonist/agonist may also be employed to preventhyper-vascular diseases, and prevent the proliferation of epitheliallens cells after extracapsular cataract surgery. Prevention of themitogenic activity of the polypeptides of the present invention may alsobe desirous in cases such as restenosis after balloon angioplasty.

[0703] The antagonist/agonist may also be employed to prevent the growthof scar tissue during wound healing.

[0704] The antagonist/agonist may also be employed to treat the diseasesdescribed herein.

[0705] Thus, the invention provides a method of treating disorders ordiseases, including but not limited to the disorders or diseases listedthroughout this application, associated with overexpression of apolynucleotide of the present invention by administering to a patient(a) an antisense molecule directed to the polynucleotide of the presentinvention, and/or (b) a ribozyme directed to the polynucleotide of thepresent invention.

[0706] Binding Peptides and Other Molecules

[0707] The invention also encompasses screening methods for identifyingpolypeptides and nonpolypeptides that bind apoptosis relatedpolypeptides, and the apoptosis related binding molecules identifiedthereby. These binding molecules are useful, for example, as agonistsand antagonists of the apoptosis related polypeptides. Such agonists andantagonists can be used, in accordance with the invention, in thetherapeutic embodiments described in detail, below.

[0708] This method comprises the steps of:

[0709] a. contacting apoptosis related polypeptides or apoptosisrelated-like polypeptides with a plurality of molecules; and

[0710] b. identifying a molecule that binds the apoptosis relatedpolypeptides or apoptosis related-like polypeptides.

[0711] The step of contacting the apoptosis related polypeptides orapoptosis related-like polypeptides with the plurality of molecules maybe effected in a number of ways. For example, one may contemplateimmobilizing the apoptosis related polypeptides or apoptosisrelated-like polypeptides on a solid support and bringing a solution ofthe plurality of molecules in contact with the immobilized apoptosisrelated polypeptides or apoptosis related-like polypeptides. Such aprocedure would be akin to an affinity chromatographic process, with theaffinity matrix being comprised of the immobilized apoptosis relatedpolypeptides or apoptosis related-like polypeptides. The moleculeshaving a selective affinity for the apoptosis related polypeptides orapoptosis related-like polypeptides can then be purified by affinityselection. The nature of the solid support, process for attachment ofthe apoptosis related polypeptides or apoptosis related-likepolypeptides to the solid support, solvent, and conditions of theaffinity isolation or selection are largely conventional and well knownto those of ordinary skill in the art.

[0712] Alternatively, one may also separate a plurality of polypeptidesinto substantially separate fractions comprising a subset of orindividual polypeptides. For instance, one can separate the plurality ofpolypeptides by gel electrophoresis, column chromatography, or likemethod known to those of ordinary skill for the separation ofpolypeptides. The individual polypeptides can also be produced by atransformed host cell in such a way as to be expressed on or about itsouter surface (e.g., a recombinant phage). Individual isolates can thenbe “probed” by the apoptosis related polypeptides or apoptosisrelated-like polypeptides, optionally in the presence of an inducershould one be required for expression, to determine if any selectiveaffinity interaction takes place between the apoptosis relatedpolypeptides or apoptosis related-like polypeptides and the individualclone. Prior to contacting the apoptosis related polypeptides orapoptosis related-like polypeptides with each fraction comprisingindividual polypeptides, the polypeptides could first be transferred toa solid support for additional convenience. Such a solid support maysimply be a piece of filter membrane, such as one made of nitrocelluloseor nylon. In this manner, positive clones could be identified from acollection of transformed host cells of an expression library, whichharbor a DNA construct encoding a polypeptide having a selectiveaffinity for apoptosis related polypeptides or apoptosis related-likepolypeptides. Furthermore, the amino acid sequence of the polypeptidehaving a selective affinity for the apoptosis related polypeptides orapoptosis related-like polypeptides can be determined directly byconventional means or the coding sequence of the DNA encoding thepolypeptide can frequently be determined more conveniently. The primarysequence can then be deduced from the corresponding DNA sequence. If theamino acid sequence is to be determined from the polypeptide itself, onemay use microsequencing techniques. The sequencing technique may includemass spectroscopy.

[0713] In certain situations, it may be desirable to wash away anyunbound apoptosis related polypeptides or apoptosis related-likepolypeptides, or altemtatively, unbound polypeptides, from a mixture ofthe apoptosis related polypeptides or apoptosis related-likepolypeptides and the plurality of polypeptides prior to attempting todetermine or to detect the presence of a selective affinity interaction.Such a wash step may be particularly desirable when the apoptosisrelated polypeptides or apoptosis related-like polypeptides or theplurality of polypeptides is bound to a solid support.

[0714] The plurality of molecules provided according to this method maybe provided by way of diversity libraries, such as random orcombinatorial peptide or nonpeptide libraries which can be screened formolecules that specifically bind apoptosis related polypeptides. Manylibraries are known in the art that can be used, e.g., chemicallysynthesized libraries, recombinant (e.g., phage display libraries), andin vitro translation-based libraries. Examples of chemically synthesizedlibraries are described in Fodor et al., 1991, Science 251:767-773;Houghten et al., 1991, Nature 354:84-86; Lam et al., 1991, Nature354:82-84; Medynski, 1994, Bio/Technology 12:709-710;Gallop et al.,1994, J. Medicinal Chemistry 37(9):1233-1251; Ohlmeyer et al., 1993,Proc. Natl. Acad. Sci. USA 90:10922-10926; Erb et al., 1994, Proc. Natl.Acad. Sci. USA 91:11422-11426; Houghten et al., 1992, Biotechniques13:412; Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA91:1614-1618; Salmon et al., 1993, Proc. Natl. Acad. Sci. USA90:11708-11712; PCT Publication No. WO 93/20242; and Brenner and Lerner,1992, Proc. Natl. Acad. Sci. USA 89:5381-5383.

[0715] Examples of phage display libraries are described in Scott andSmith, 1990, Science 249:386-390; Devlin et al., 1990, Science,249:404-406; Christian, R. B., et al., 1992, J. Mol. Biol. 227:711-718);Lenstra, 1992, J. Immunol. Meth. 152:149-157; Kay et al., 1993, Gene128:59-65; and PCT Publication No. WO 94/18318 dated Aug. 18, 1994.

[0716] In vitro translation-based libraries include but are not limitedto those described in PCT Publication No. WO 91/05058 dated Apr. 18,1991; and Mattheakis et al., 1994, Proc. Natl. Acad. Sci. USA91:9022-9026.

[0717] By way of examples of nonpeptide libraries, a benzodiazepinelibrary (see e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA91:4708-4712) can be adapted for use. Peptoid libraries (Simon et al.,1992, Proc. Natl. Acad. Sci. USA 89:9367-9371) can also be used. Anotherexample of a library that can be used, in which the amidefunctionalities in peptides have been permethylated to generate achemically transformed combinatorial library, is described by Ostresh etal. (1994, Proc. Natl. Acad. Sci. USA 91:11138-11142).

[0718] The variety of non-peptide libraries that are useful in thepresent invention is great. For example, Ecker and Crooke, 1995,Bio/Technology 13:351-360 list benzodiazepines, hydantoins,piperazinediones, biphenyls, sugar analogs, beta-mercaptoketones,arylacetic acids, acylpiperidines, benzopyrans, cubanes, xanthines,aminimides, and oxazolones as among the chemical species that form thebasis of various libraries.

[0719] Non-peptide libraries can be classified broadly into two types:decorated monomers and oligomers. Decorated monomer libraries employ arelatively simple scaffold structure upon which a variety functionalgroups is added. Often the scaffold will be a molecule with a knownuseful pharmacological activity. For example, the scaffold might be thebenzodiazepine structure.

[0720] Non-peptide oligomer libraries utilize a large number of monomersthat are assembled together in ways that create new shapes that dependon the order of the monomers. Among the monomer units that have beenused are carbamates, pyrrolinones, and morpholinos. Peptoids,peptide-like oligomers in which the side chain is attached to the alphaamino group rather than the alpha carbon, form the basis of anotherversion of non-peptide oligomer libraries. The first non-peptideoligomer libraries utilized a single type of monomer and thus containeda repeating backbone. Recent libraries have utilized more than onemonomer, giving the libraries added flexibility.

[0721] Screening the libraries can be accomplished by any of a varietyof commonly known methods. See, e.g., the following references, whichdisclose screening of peptide libraries: Parmley and Smith, 1989, Adv.Exp. Med. Biol. 251:215-218; Scott and Smith, 1990, Science 249:386-390;Fowlkes et al., 1992; BioTechniques 13:422-427; Oldenburg et al., 1992,Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et al., 1994, Cell76:933-945; Staudt et al., 1988, Science 241:577-580; Bock et al., 1992,Nature 355:564-566; Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA89:6988-6992; Ellington et al., 1992, Nature 355:850-852; U.S. Pat. Nos.5,096,815, 5,223,409, and 5,198,346, all to Ladner et al.; Rebar andPabo, 1993, Science 263:671-673; and CT Publication No. WO 94/18318.

[0722] In a specific embodiment, screening to identify a molecule thatbinds apoptosis related polypeptides can be carried out by contactingthe library members with a apoptosis related polypeptides or apoptosisrelated-like polypeptides immobilized on a solid phase and harvestingthose library members that bind to the apoptosis related polypeptides orapoptosis related-like polypeptides. Examples of such screening methods,termed “panning” techniques are described by way of example in Parmleyand Smith, 1988, Gene 73:305-318; Fowlkes et al., 1992, BioTechniques13:422-427; PCT Publication No. WO 94/18318; and in references citedherein.

[0723] In another embodiment, the two-hybrid system for selectinginteracting proteins in yeast (Fields and Song, 1989, Nature340:245-246; Chien et al., 1991, Proc. Natl. Acad. Sci. USA88:9578-9582) can be used to identify molecules that specifically bindto apoptosis related polypeptides or apoptosis related-likepolypeptides.

[0724] Where the apoptosis related binding molecule is a polypeptide,the polypeptide can be conveniently selected from any peptide library,including random peptide libraries, combinatorial peptide libraries, orbiased peptide libraries. The term “biased” is used herein to mean thatthe method of generating the library is manipulated so as to restrictone or more parameters that govern the diversity of the resultingcollection of molecules, in this case peptides.

[0725] Thus, a truly random peptide library would generate a collectionof peptides in which the probability of finding a particular amino acidat a given position of the peptide is the same for all 20 amino acids. Abias can be introduced into the library, however, by specifying, forexample, that a lysine occur every fifth amino acid or that positions 4,8, and 9 of a decapeptide library be fixed to include only arginine.Clearly, many types of biases can be contemplated, and the presentinvention is not restricted to any particular bias. Furthermore, thepresent invention contemplates specific types of peptide libraries, suchas phage displayed peptide libraries and those that utilize a DNAconstruct comprising a lambda phage vector with a DNA insert.

[0726] As mentioned above, in the case of a apoptosis related bindingmolecule that is a polypeptide, the polypeptide may have about 6 to lessthan about 60 amino acid residues, preferably about 6 to about 10 aminoacid residues, and most preferably, about 6 to about 22 amino acids. Inanother embodiment, a apoptosis related binding polypeptide has in therange of 15-100 amino acids, or 20-50 amino acids.

[0727] The selected apoptosis related binding polypeptide can beobtained by chemical synthesis or recombinant expression.

[0728] Other Activities

[0729] A polypeptide, polynucleotide, agonist, or antagonist of thepresent invention, as a result of the ability to stimulate vascularendothelial cell growth, may be employed in treatment for stimulatingre-vascularization of ischemic tissues due to various disease conditionssuch as thrombosis, arteriosclerosis, and other cardiovascularconditions. The polypeptide, polynucleotide, agonist, or antagonist ofthe present invention may also be employed to stimulate angiogenesis andlimb regeneration, as discussed above.

[0730] A polypeptide, polynucleotide, agonist, or antagonist of thepresent invention may also be employed for treating wounds due toinjuries, bums, post-operative tissue repair, and ulcers since they aremitogenic to various cells of different origins, such as fibroblastcells and skeletal muscle cells, and therefore, facilitate the repair orreplacement of damaged or diseased tissue.

[0731] A polypeptide, polynucleotide, agonist, or antagonist of thepresent invention may also be employed stimulate neuronal growth and totreat and prevent neuronal damage which occurs in certain neuronaldisorders or neuro-degenerative conditions such as Alzheimer's disease,Parkinson's disease, and AIDS-related complex. A polypeptide,polynucleotide, agonist, or antagonist of the present invention may havethe ability to stimulate chondrocyte growth, therefore, they may beemployed to enhance bone and periodontal regeneration and aid in tissuetransplants or bone grafts.

[0732] A polypeptide, polynucleotide, agonist, or antagonist of thepresent invention may be also be employed to prevent skin aging due tosunburn by stimulating keratinocyte growth.

[0733] A polypeptide, polynucleotide, agonist, or antagonist of thepresent invention may also be employed for preventing hair loss, sinceFGF family members activate hair-forming cells and promotes melanocytegrowth. Along the same lines, a polypeptide, polynucleotide, agonist, orantagonist of the present invention may be employed to stimulate growthand differentiation of hematopoietic cells and bone marrow cells whenused in combination with other cytokines.

[0734] A polypeptide, polynucleotide, agonist, or antagonist of thepresent invention may also be employed to maintain organs beforetransplantation or for supporting cell culture of primary tissues. Apolypeptide, polynucleotide, agonist, or antagonist of the presentinvention may also be employed for inducing tissue of mesodermal originto differentiate in early embryos.

[0735] A polypeptide, polynucleotide, agonist, or antagonist of thepresent invention may also increase or decrease the differentiation orproliferation of embryonic stem cells, besides, as discussed above,hematopoietic lineage.

[0736] A polypeptide, polynucleotide, agonist, or antagonist of thepresent invention may also be used to modulate mammaliancharacteristics, such as body height, weight, hair color, eye color,skin, percentage of adipose tissue, pigmentation, size, and shape (e.g.,cosmetic surgery). Similarly, a polypeptide, polynucleotide, agonist, orantagonist of the present invention may be used to modulate mammalianmetabolism affecting catabolism, anabolism, processing, utilization, andstorage of energy.

[0737] A polypeptide, polynucleotide, agonist, or antagonist of thepresent invention may be used to change a mammal's mental state orphysical state by influencing biorhythms, caricadic rhythms, depression(including depressive disorders), tendency for violence, tolerance forpain, reproductive capabilities (preferably by Activin or Inhibin-likeactivity), hormonal or endocrine levels, appetite, libido, memory,stress, or other cognitive qualities.

[0738] A polypeptide, polynucleotide, agonist, or antagonist of thepresent invention may also be used as a food additive or preservative,such as to increase or decrease storage capabilities, fat content,lipid, protein, carbohydrate, vitamins, minerals, cofactors or othernutritional components.

[0739] The above-recited applications have uses in a wide variety ofhosts. Such hosts include, but are not limited to, human, murine,rabbit, goat, guinea pig, camel, horse, mouse, rat, hamster, pig,micro-pig, chicken, goat, cow, sheep, dog, cat, non-human primate, andhuman. In specific embodiments, the host is a mouse, rabbit, goat,guinea pig, chicken, rat, hamster, pig, sheep, dog or cat. In preferredembodiments, the host is a mammal. In most preferred embodiments, thehost is a human.

[0740] Other Preferred Embodiments

[0741] Other preferred embodiments of the claimed invention include anisolated nucleic acid molecule comprising a nucleotide sequence which isat least 95% identical to a sequence of at least about 50 contiguousnucleotides in the nucleotide sequence of SEQ ID NO:X or thecomplementary strand thereto, and/or cDNA plasmid:Z.

[0742] Also preferred is a nucleic acid molecule wherein said sequenceof contiguous nucleotides is included in the nucleotide sequence of SEQID NO:X in the range of positions identified for SEQ ID NO:X in Table 1.

[0743] Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a sequence of atleast about 150 contiguous nucleotides in the nucleotide sequence of SEQID NO:X or the complementary strand thereto, and/or cDNA plasmid:Z.

[0744] Further preferred is an isolated nucleic acid molecule comprisinga nucleotide sequence which is at least 95% identical to a sequence ofat least about 500 contiguous nucleotides in the nucleotide sequence ofSEQ ID NO:X or the complementary strand thereto, and/or cDNA plasmid:Z.

[0745] A further preferred embodiment is a nucleic acid moleculecomprising a nucleotide sequence which is at least 95% identical to thenucleotide sequence of SEQ ID NO:X in the range of positions identifiedfor SEQ ID NO:X in Table 1.

[0746] A further preferred embodiment is an isolated nucleic acidmolecule comprising a nucleotide sequence which is at least 95%identical to the complete nucleotide sequence of SEQ ID NO:X or thecomplementary strand thereto, and/or cDNA plasmid:Z.

[0747] Also preferred is an isolated nucleic acid molecule whichhybridizes under stringent hybridization conditions to a nucleic acidmolecule comprising a nucleotide sequence of SEQ ID NO:X or thecomplementary strand thereto and/or cDNA plasmid:Z, wherein said nucleicacid molecule which hybridizes does not hybridize under stringenthybridization conditions to a nucleic acid molecule having a nucleotidesequence consisting of only A residues or of only T residues.

[0748] Also preferred is a composition of matter comprising a DNAmolecule which comprises cDNA plasmid:Z.

[0749] Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a sequence of atleast 50 contiguous nucleotides in the nucleotide sequence of cDNAplasmid:Z.

[0750] Also preferred is an isolated nucleic acid molecule, wherein saidsequence of at least 50 contiguous nucleotides is included in thenucleotide sequence of an open reading frame sequence encoded by cDNAplasmid:Z.

[0751] Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to sequence of atleast 150 contiguous nucleotides in the nucleotide sequence encoded bycDNA plasmid:Z.

[0752] A further preferred embodiment is an isolated nucleic acidmolecule comprising a nucleotide sequence which is at least 95%identical to sequence of at least 500 contiguous nucleotides in thenucleotide sequence encoded by cDNA plasmid:Z.

[0753] A further preferred embodiment is an isolated nucleic acidmolecule comprising a nucleotide sequence which is at least 95%identical to the complete nucleotide sequence encoded by cDNA plasmid:Z.

[0754] A further preferred embodiment is a method for detecting in abiological sample a nucleic acid molecule comprising a nucleotidesequence which is at least 95% identical to a sequence of at least 50contiguous nucleotides in a sequence selected from the group consistingof: a nucleotide sequence of SEQ ID NO:X or the complementary strandthereto and a nucleotide sequence encoded by cDNA plasmid:Z; whichmethod comprises a step of comparing a nucleotide sequence of at leastone nucleic acid molecule in said sample with a sequence selected fromsaid group and determining whether the sequence of said nucleic acidmolecule in said sample is at least 95% identical to said selectedsequence.

[0755] Also preferred is the above method wherein said step of comparingsequences comprises determining the extent of nucleic acid hybridizationbetween nucleic acid molecules in said sample and a nucleic acidmolecule comprising said sequence selected from said group. Similarly,also preferred is the above method wherein said step of comparingsequences is performed by comparing the nucleotide sequence determinedfrom a nucleic acid molecule in said sample with said sequence selectedfrom said group. The nucleic acid molecules can comprise DNA moleculesor RNA molecules.

[0756] A further preferred embodiment is a method for identifying thespecies, tissue or cell type of a biological sample which methodcomprises a step of detecting nucleic acid molecules in said sample, ifany, comprising a nucleotide sequence that is at least 95% identical toa sequence of at least 50 contiguous nucleotides in a sequence selectedfrom the group consisting of: a nucleotide sequence of SEQ ID NO:X orthe complementary strand thereto and a nucleotide sequence encoded bycDNA plasmid:Z.

[0757] The method for identifying the species, tissue or cell type of abiological sample can comprise a step of detecting nucleic acidmolecules comprising a nucleotide sequence in a panel of at least twonucleotide sequences, wherein at least one sequence in said panel is atleast 95% identical to a sequence of at least 50 contiguous nucleotidesin a sequence selected from said group.

[0758] Also preferred is a method for diagnosing in a subject apathological condition associated with abnormal structure or expressionof a nucleotide sequence of SEQ ID NO:X or the complementary strandthereto or cDNA plasmid:Z which encodes a protein, wherein the methodcomprises a step of detecting in a biological sample obtained from saidsubject nucleic acid molecules, if any, comprising a nucleotide sequencethat is at least 95% identical to a sequence of at least 50 contiguousnucleotides in a sequence selected from the group consisting of: anucleotide sequence of SEQ ID NO:X or the complementary strand theretoand a nucleotide sequence of cDNA plasmid:Z.

[0759] The method for diagnosing a pathological condition can comprise astep of detecting nucleic acid molecules comprising a nucleotidesequence in a panel of at least two nucleotide sequences, wherein atleast one sequence in said panel is at least 95% identical to a sequenceof at least 50 contiguous nucleotides in a sequence selected from saidgroup.

[0760] Also preferred is a composition of matter comprising isolatednucleic acid molecules wherein the nucleotide sequences of said nucleicacid molecules comprise a panel of at least two nucleotide sequences,wherein at least one sequence in said panel is at least 95% identical toa sequence of at least 50 contiguous nucleotides in a sequence selectedfrom the group consisting of: a nucleotide sequence of SEQ ID NO:X orthe complementary strand thereto and a nucleotide sequence encoded bycDNA plasmid:Z. The nucleic acid molecules can comprise DNA molecules orRNA molecules.

[0761] Also preferred is an isolated polypeptide comprising an aminoacid sequence at least 90% identical to a sequence of at least about 10contiguous amino acids in the polypeptide sequence of SEQ ID NO:Y; apolypeptide encoded by SEQ ID NO:X or the complementary strand theretoand/or a polypeptide encoded by cDNA plasmid:Z.

[0762] Also preferred is an isolated polypeptide comprising an aminoacid sequence at least 95% identical to a sequence of at least about 30contiguous amino acids in the amino acid sequence of SEQ ID NO:Y; apolypeptide encoded by SEQ ID NO:X or the complementary strand theretoand/or a polypeptide encoded by cDNA plasmid:Z.

[0763] Further preferred is an isolated polypeptide comprising an aminoacid sequence at least 95% identical to a sequence of at least about 100contiguous amino acids in the amino acid sequence of SEQ ID NO:Y; apolypeptide encoded by SEQ ID NO:X or the complementary strand theretoand/or a polypeptide encoded by cDNA plasmid:Z.

[0764] Further preferred is an isolated polypeptide comprising an aminoacid sequence at least 95% identical to the complete amino acid sequenceof SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or thecomplementary strand thereto and/or a polypeptide encoded by cDNAplasmid:Z.

[0765] Further preferred is an isolated polypeptide comprising an aminoacid sequence at least 90% identical to a sequence of at least about 10contiguous amino acids in the complete amino acid sequence of apolypeptide encoded by cDNA plasmid:Z

[0766] Also preferred is a polypeptide wherein said sequence ofcontiguous amino acids is included in the amino acid sequence of aportion of said polypeptide encoded by cDNA plasmid:Z; a polypeptideencoded by SEQ ID NO:X or the complementary strand thereto and/or thepolypeptide sequence of SEQ ID NO:Y.

[0767] Also preferred is an isolated polypeptide comprising an aminoacid sequence at least 95% identical to a sequence of at least about 30contiguous amino acids in the amino acid sequence of a polypeptideencoded by cDNA plasmid:Z.

[0768] Also preferred is an isolated polypeptide comprising an aminoacid sequence at least 95% identical to a sequence of at least about 100contiguous amino acids in the amino acid sequence of a polypeptideencoded by cDNA plasmid:Z.

[0769] Also preferred is an isolated polypeptide comprising an aminoacid sequence at least 95% identical to the amino acid sequence of apolypeptide encoded by cDNA plasmid:Z.

[0770] Further preferred is an isolated antibody which bindsspecifically to a polypeptide comprising an amino acid sequence that isat least 90% identical to a sequence of at least 10 contiguous aminoacids in a sequence selected from the group consisting of: a polypeptidesequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or thecomplementary strand thereto and a polypeptide encoded by cDNAplasmid:Z.

[0771] Further preferred is a method for detecting in a biologicalsample a polypeptide comprising an amino acid sequence which is at least90% identical to a sequence of at least 10 contiguous amino acids in asequence selected from the group consisting of: a polypeptide sequenceof SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or thecomplementary strand thereto and a polypeptide encoded by cDNAplasmid:Z; which method comprises a step of comparing an amino acidsequence of at least one polypeptide molecule in said sample with asequence selected from said group and determining whether the sequenceof said polypeptide molecule in said sample is at least 90% identical tosaid sequence of at least 10 contiguous amino acids.

[0772] Also preferred is the above method wherein said step of comparingan amino acid sequence of at least one polypeptide molecule in saidsample with a sequence selected from said group comprises determiningthe extent of specific binding of polypeptides in said sample to anantibody which binds specifically to a polypeptide comprising an aminoacid sequence that is at least 90% identical to a sequence of at least10 contiguous amino acids in a sequence selected from the groupconsisting of: a polypeptide sequence of SEQ ID NO:Y; a polypeptideencoded by SEQ ID NO:X or the complementary strand thereto and apolypeptide encoded by cDNA plasmid:Z.

[0773] Also preferred is the above method wherein said step of comparingsequences is performed by comparing the amino acid sequence determinedfrom a polypeptide molecule in said sample with said sequence selectedfrom said group.

[0774] Also preferred is a method for identifying the species, tissue orcell type of a biological sample which method comprises a step ofdetecting polypeptide molecules in said sample, if any, comprising anamino acid sequence that is at least 90% identical to a sequence of atleast 10 contiguous amino acids in a sequence selected from the groupconsisting of: polypeptide sequence of SEQ ID NO:Y; a polypeptideencoded by SEQ ID NO:X or the complementary strand thereto and apolypeptide encoded by cDNA plasmid:Z.

[0775] Also preferred is the above method for identifying the species,tissue or cell type of a biological sample, which method comprises astep of detecting polypeptide molecules comprising an amino acidsequence in a panel of at least two amino acid sequences, wherein atleast one sequence in said panel is at least 90% identical to a sequenceof at least 10 contiguous amino acids in a sequence selected from theabove group.

[0776] Also preferred is a method for diagnosing in a subject apathological condition associated with abnormal structure or expressionof a nucleic acid sequence identified in Table 1 encoding a polypeptide,which method comprises a step of detecting in a biological sampleobtained from said subject polypeptide molecules comprising an aminoacid sequence in a panel of at least two amino acid sequences, whereinat least one sequence in said panel is at least 90% identical to asequence of at least 10 contiguous amino acids in a sequence selectedfrom the group consisting of: polypeptide sequence of SEQ ID NO:Y; apolypeptide encoded by SEQ ID NO:X or the complementary strand theretoand a polypeptide encoded by cDNA plasmid:Z.

[0777] In any of these methods, the step of detecting said polypeptidemolecules includes using an antibody.

[0778] Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a nucleotidesequence encoding a polypeptide wherein said polypeptide comprises anamino acid sequence that is at least 90% identical to a sequence of atleast 10 contiguous amino acids in a sequence selected from the groupconsisting of: polypeptide sequence of SEQ ID NO:Y; a polypeptideencoded by SEQ ID NO:X or the complementary strand thereto and apolypeptide encoded by cDNA plasmid:Z.

[0779] Also preferred is an isolated nucleic acid molecule, wherein saidnucleotide sequence encoding a polypeptide has been optimized forexpression of said polypeptide in a prokaryotic host.

[0780] Also preferred is an isolated nucleic acid molecule, wherein saidpolypeptide comprises an amino acid sequence selected from the groupconsisting of: polypeptide sequence of SEQ ID NO:Y; a polypeptideencoded by SEQ ID NO:X or the complementary strand thereto and apolypeptide encoded by cDNA plasmid:Z.

[0781] Further preferred is a method of making a recombinant vectorcomprising inserting any of the above isolated nucleic acid moleculeinto a vector. Also preferred is the recombinant vector produced by thismethod. Also preferred is a method of making a recombinant host cellcomprising introducing the vector into a host cell, as well as therecombinant host cell produced by this method.

[0782] Also preferred is a method of making an isolated polypeptidecomprising culturing this recombinant host cell under conditions suchthat said polypeptide is expressed and recovering said polypeptide. Alsopreferred is this method of making an isolated polypeptide, wherein saidrecombinant host cell is a eukaryotic cell and said polypeptide is ahuman protein comprising an amino acid sequence selected from the groupconsisting of: polypeptide sequence of SEQ ID NO:Y; a polypeptideencoded by SEQ ID NO:X or the complementary strand thereto and apolypeptide encoded by cDNA plasmid:Z. The isolated polypeptide producedby this method is also preferred.

[0783] Also preferred is a method of treatment of an individual in needof an increased level of a protein activity, which method comprisesadministering to such an individual a Therapeutic comprising an amountof an isolated polypeptide, polynucleotide, immunogenic fragment oranalogue thereof, binding agent, antibody, or antigen binding fragmentof the claimed invention effective to increase the level of said proteinactivity in said individual.

[0784] Also preferred is a method of treatment of an individual in needof a decreased level of a protein activity, which method comprisedadministering to such an individual a Therapeutic comprising an amountof an isolated polypeptide, polynucleotide, immunogenic fragment oranalogue thereof, binding agent, antibody, or antigen binding fragmentof the claimed invention effective to decrease the level of said proteinactivity in said individual.

[0785] In specific embodiments of the invention, for each “Contig ID”listed in the fourth column of Table 2, preferably excluded are one ormore polynucleotides comprising, or alternatively consisting of, anucleotide sequence referenced in the fifth column of Table 2 anddescribed by the general formula of a-b, whereas a and b are uniquelydetermined for the corresponding SEQ ID NO:X referred to in column 3 ofTable 2. Further specific embodiments are directed to polynucleotidesequences excluding one, two, three, four, or more of the specificpolynucleotide sequences referred to in the fifth column of Table 2. Inno way is this listing meant to encompass all of the sequences which maybe excluded by the general formula, it is just a representative example.All references available through these accessions are herebyincorporated by reference in their entirety. TABLE 2 NT SEQ ID cDNA NO:Contig Gene No. Clone ID X ID Public Accession Numbers 1 HLDOK36 2846025 AW245401, AA662107, AI523949, AW245758, AI031817, AA725300,AI359207, AW270125, AA293413, AI090434, AA568269, AW013988, AA708767,AA682427, AI376689, AI033528, AW449244, C01335, AW263988, AI343327,AI360743, T50230, AI992119, AA908655, AA318766, T50243, AA635978,AW204989, AA830678, AA047668, AA748433, AA383495, AI635643, AA862542,F35595, AA218681, AI358311, AA090354, AI432940, AW050934, AW362290,AI636445, AW075351, AI800433, AL135661, AI349957, AL044207, AI800453,AI343112, AI349598, AI345735, AW080079, AW268253, AW148320, AI281837,AL036980, AW089572, AW129171, AI597750, AI290154, AW149851, AI282281,AW090013, AI869367, AI340582, AW075413, AI500077, AI567612, AI572787,AW074993, AW302992, AI538790, AI500659, AL119457, AI312152, AW080279,AI571861, AI349614, AI440426, AI925156, AI801544, AI309401, AW075084,AI784252, AI270707, AI348897, AI307708, AI349937, AI567351, AI439089,AI439717, AI862144, AI758437, AI590128, AL036403, AI950664, AI282655,AW169653, AI634224, AL040243, AI279984, AI281779, AW193635, AI475134,AI620639, AI499463, AW071349, AI684265, AI349004, AI862142, AL036146,AW268220, AI445165, AI568855, AW301300, AW075207, AI349256, AA508692,AI343037, AI520862, AI648684, AL038778, AI349645, AI334884, AI632033,AL121014, AI569583, AI497733, AW274192, AI313352, AW301409, AI560099,AI857296, AI633073, AI312428, AI580927, AI274541, AW071417, AA225339,AI627893, AI828818, AI818206, AI436456, AI273142, AI571133, AI609190,AWl51485, AW008048, AI281773, AA470491, AI636183, AI636585, AI572569,AI819970, AI919058, AI274508, AI564247, AI699857, AW149287, AW183621,AW068845, AI783504, AI824764, AW302965, AI436644, AW074869, AW263453,AI680388, AI564992, AI269862, AI536638, AI702068, AI349226, AI627360,AI249257, AI491852, AI952360, AI249323, AI273048, AL043326, AW118512,AW131954, AI653836, AL036396, AW196141, AI612920, AI439478, AI269205,AI678989, AW104724, AI554484, AI349933, AI682841, AI624206, AI610756,AI811344, AL036361, AW087445, AI912866, AI571551, AI690312, AI275175,AI702406, AI637584, AI340603, AI570384, AI538716, AI690490, AW002342,AI475451, AI569616, AI872074, AI872711, AI702433, AW301505, AI224992,AI799199, AI679764, AI554427, AW082040, AI1815855, AW269097, AI926790,AI564719, AI653541, AI269696, AI889376, AI874109, AI499146, AI868831,AW103371, AI524671, AI521012, AI591073, AI633419, AI921248, AI307543,AI498579, AI590120, AI866002, AI619502, AI571909, AI433976, AI802542,AI866100, AI744923, AI922901, AI828731, AI917253, I48979, Y11587,I89947, I89931, AF090943, AF113699, AF113694, AF118064, AL049314,A08916, AF118070, A08913, L31396, L31397, AL049452, AF113013, AJ242859,AL110221, AL080124, U42766, AL133557, AL122093, AL050393, AF113691,AB019565, AF078844, AF113690, AF113677, AL137557, AL133093, Y11254,AL122050, AF111851, AL117460, AL050149, AL050116, AF125949, AL050146,AL133606, AF113689, AL122123, S68736, X84990, AF090900, AL133565,AL133640, AF113676, AF158248, AL050108, S78214, AF090903, AL080060,AF090896, AF091084, AF113019, E03348, AF090934, AL110196, AL049466,AR059958, I48978, AL133075, AL117457, AL133016, AF125948, AL080137,AF090901, AL137527, X63574, AL122121, AF106862, E07361, A93016,AF017152, AL133080, AF146568, AL049938, AL050277, AL137459, AL117394,X82434, AL110225, AF104032, AJ000937, AL096744, U91329, AL050138,AF079765, I49625, AF017437, AL137283, Y16645, AL049464, AL133560,AL117585, E02349, AR011880, AL137550, AJ238278, A65341, U00763, A08910,AL049300, AF177401, AF067728, A08912, AF097996, AL049430, E07108,AL117583, AL117435, AL049382, A58524, A58523, A08909, AL137521,AF118094, Z82022, AF183393, I03321, AL122098, AL137648, X96540, U72620,AL050024, X70685, A77033, A77035, AL137463, X72889, AL137271, AL137538,AL080127, U80742, AL133113, AI2297, U35846, I33392, A03736, AL122110,AL049283, AF087943, X93495, I09360, X65873, X98834, S61953, AL110197,I17767, AF061943, AL080159, E08263, E08264, AF026124, U67958, AC006336,I42402, Y09972, AL137560, AL133568, AL122049, AL133072, AR038969,E15569, AL133014, Y07905, AF095901, AL133098, AJ012755, AL137523,I66342, AR054984, AF111112, I26207, AL133077, M30514, I00734, AF026816,AF119337, AL110280, A93350, E00617, E00717, E00778, A08911, AR000496,U39656, Z37987, AL137556, AL137526, AL137429, AC004093, AF061573,U68387, AL133104, AF003737, A45787, AF000145, AL050172, Y14314,AF106827, AF057300, AF057299, AR013797, A90832, AL122111, U58996,A07647, AF079763, X83508, AF100931, Z72491, AF153205, AF185576, E08631,U78525, AR038854, AL137292, AF162270, AL133067, E04233, AL080074,AL117649, U96683, AL117440, AL137476, X87582, AF210052, L13297,AC006371, E05822, AF051325, L30117, AL137656, AL050092, AC002467,AL133081, AL137533, AJ006417, X92070, and AF091512. 2 HDPBW68 3 847093AI797914, AA232727, AI264354, AA242826, AI42ll52, AI373844, AI693559,AA293798, AA242961, AI681069, AA987481, AA253496, AA394280, AA865918,AW193319, AA699441, AA534330, AI246675, AI690035, AI921391, AI696791,AI696792, AI962498, AA478182, AA845215, R02588, AA501984, AA253392,AA975909, AI141321, AI359321, R02707, AI370136, AI424757, AA236520,AA065210, AI369930, AA064845, AI217878, AI470976, AI640699, AF113925,AF126484, AF149774, AC006027, AF149773, and AC005154. 2 HDPBW68 10835653 3 HHEFO24 4 846324 AW410791, AI910444, AW410792, AW135479,AI609413, AI733753, AI479543, AA052898, AA053071, AA599894, AI651518,AI262133, AW206814, AW242057, N77940, AI741979, AA026620, AA026732,AI692702, AI690860, W06897, T84089, AW449583, AW206187, AA310890,AA047315, AI492080, AA334702, AA310840, AI572152, AA047316, T83444,AI525358, AW407496, H99721, AA334684, AA932181, AA236177, H63078,AA100344, and AB020694. 4 HEGAL46 5 839584 AI951905, AI935307, AW293446,AI694308, AA631O67, AA714504, N39432, AW450043, AI193673, AA642327,N50632, AI674982, AA507436, AA826605, AA598930, R13288, N55392, R45988,and AA377586. 5 HFOYCO2 6 775677 AA203338, AI693169, AW003902, AL135076,AI597610, AA716206, AI479987, AA582941, AI692611, AA485609, AI889044,D83867, N27871, AW080978, AI568020, AW316656, AI201938, AAI55950,AA490228, D55820, AI299131, H18712, AW069403, N40648, AA236975,AA305709, AA765761, AAI92998, AA946944, N33286, AA393877, AA252795,H59030, AA862222, T63672, AA261817, R52868, Z43502, AA211032, AI039732,AI198010, T09360, T17275, AA336378, AA360198, H59029, D82324, AA730251,AA545761, D55791, AAI93049, AW071584, T33758, R82391, N20127, R66449,AI880244, T32936, AA453221, AAI55905, AA235913, AI057001, N84480,T10639, AI458373, AA974036, AI884730, D55965, AA320670, AW403754,AI204172, AI873966, AA262514, Z42774, W01410, T11193, AA723391,AA887396, AA318022, M85632, AI470045, AI872411, AI289832, AA894810,AI052412, F02862, AA707555, AA485443, AA716192, AF077599, and AL080105.6 HDABV82 7 828174 AW069306, AI743175, AI803970, AI811472, AI027704,AA099277, AI168623, AI276150, AI824726, AA213703, AW243339, AA483707,AA213668, AI653168, AA371310, R38844, AI434885, AA342147, AA342146,AA828028, AI581083, AA099276, AB023172, AP000245, AP000127, andAP000205. 7 HSVAF16 8 845403 AW027073, AL038494, AI888327, AI361046,AI953282, AW385192, AW385191, AW072643, P1675185, AW005989, AI193532,AI688965, AA568251, AA843521, AA766104, AI379780, AI934841, AI479734,N40017, AA588815, N27282, AA872441, AW194363, AA602586, AW305055,AA557627, AA854085, AA807817, AI357691, AA593135, AI948655, AI457174,W86872, AW082605, AA493327, W86823, AI631373, T51002, AA730909,AA507175, AA259062, AL038493, AA844181, AI280762, AA312467, AW105578,AL038837, AW371305, AL039074, AL039564, AL039128, AL039109, AL039108,AL037051, AL038531, AL039659, T39725, AL039625, AL039648, AL039629,AL039678, AL040992, AA844410, AL039156, AL037726, AL045337, AL042909,AL039386, AL039423, AL036973, AL044407, AL039410, AL045353, AL036725,AL039150, AL045341, AL044530, AA249773, AL039538, AL039566, AL039509,AL043422, AA259061, AL036196, AL039924, AL045794, T24119, T24112,AL037639, AL038025, AA323906, AL038821, AL037615, AL036767, AL043445,AL043423, AW013814, AL043441, AL039085, H00069, AL037526, AL036679,T23947, AI927577, AL036924, T02921, AA938071, AL036418, AL037027,AL036117, AL036733, AW451070, AL036158, AI535983, AL036765, AL036268,AL036964, AL036238, AI535783, D51250, AL037601, AL036998, AL037082,AL038851, R47228, AW452756, AL036190, AL037047, AL037643, AL037178,AL036191, D80253, AL037021, AL036167, AL037049, AL037085, AL037054,Z99396, AL037177, AL036227, D80043, AL036133, D59275, AL037124, D80219,AL036163, D59787, AL036207, AL037600, AL036132, AL036914, AL036139,T23659, D80227, AL036152, AL037679, AL036900, AL036228, D80240, D80134,AL036174, D51423, T11051, AL048425, AL036150, T48598, D80210, AL036858,D59619, AL036808, AL036953, AL037077, AA514190, G14227, AL037569,AW450376, D80193, AL037081, D80196, Z25782, D80391, AL038043, D80168,D59927, AL038447, D80949, AI557751, D80366, H00072, AL037002, AL119457,AL036630, AL119324, C75259, AL119399, D80045, AL042544, D81026,AA631969, AW135155, C14014, Z25783, AL043152, AA894816, AW392670,D59889, AL079794, AL036229, AL043168, AL037016, C14389, T11417, U46347,AI431323, AL036241, AA835947, D80038, D80022, AW451416, AL042866,AL041587, AI309306, AW025279, AW384394, AL119443, AI873638, X68127,A85477, AR025207, AR017907, A85396, AR062871, A25909, A84772, A84776,A84773, A84775, AR062872, A84774, AR062873, AR067731, AR037157,AR067732, A86792, A58522, A91750, I18371, A20702, A20700, A43189,A43188, AF118808, X73004, Z96142, A44171, V00745, AR036903, AI1245,I19517, A76773, E13740, A22413, I13349, A35536, A35537, A02135, A02136,A04663, A04664, I01992, I08051, A95051, AI0361, AR036905, A95117,AR031374, A49700, ARO31375, A58521, AJ244003, AJ244004, AR020969,A38214, I56772, I95540, AR018924, A63067, A51047, A63064, AR018923,A48774, A63072, A48775, AR068507, AR068506, AR015960, AR000007,AR015961, I19516, E03165, AI8053, I06859, AR043602, A23334, A75888,I70384, AR043603, A60111, A23633, A23998, AR043601, A02712, A95052,AI8050, AR007512, A98767, A93963, A93964, I63120, I60241, I60242,AR054109, A58524, A58523, A64081, A24783, A24782, I03343, A81878,A97211, AR022240, A02710, E12615, AR035193, A92133, E14304, A07700,AI3392, AI3393, A27396, AR027100, I28266, I21869, A49045, E16678,E16636, A93016, A92636, A82653, D28584, I25027, I26929, I44515, I26928,I26930, I26927, A58525, E02221, E01614, E13364, A67220, A70040,AR038762, I49890, I44516, AF156296, AR000006, U87250, AR035975,AR035974, AR035977, AR035976, AR035978, A51384, A58526, A91753, I00079,E16590, AF156294, AJ230933, AJ244005, Y11923, Y11926, AR008430, I00074,AI5078, I03665, I03664, D88984, I66494, I66495, I66498, I66497, I66496,I66486, I66487, A91965, E00523, AR038286, I25041, I92483, I00077,D34614, A20701, AF156303, AR027069, A52326, ABO12117, A04710, D14548,X13220, AF156299, I07429, I168636, A97221, AF156304, AF156302, AF019720,AR066482, AR028564, AR060673, AR060676, A49428, AI8722, S70644, M32676,AF096810, E06034, A91754, I69350, A08457, A08458, S65373, X58217,AI3038, A29289, Y17188, A60957, A00782, A02741, AI4595, AI8755, A25856,I12245, A49695, A49696, I84554, I84553, AF096793, A60968, A60985,A60990, A60987, D44443, AB007195, X15418, AF130655, AI0363, X73003,I08250, E04616, AR064706, S78798, X16234, A80951, I19525, I40851,A60983, I07888, AF156300, Y11920, I03663, S83538, and Y11449. 8 HSIFO619 859875 AW194103, AA570483, AW247071, AL134627, AA532739, AI950034,AI831065, AI791597, AW025209, AI733101, AW072315, AW088486, AW250423,AW027992, AI872396, AI822039, AI978692, AA526905, AA004890, AI521711,AI859462, AI589001, AW062708, AI149487, AA932997, AI709015, AW269349,AW269185, AI140101, AA725306, AW002658, AA481316, AI554225, AA600310,AI352315, AA651838, AAI34415, AI635661, AI431710, AW235534, AI284946,AI753373, AA705577, H20713, AW269158, AI983743, AAI34414, AI248592,AI942429, AI873092, AA913640, AI933816, AI633977, H15546, AI360370,G04532, AA004615, AW401864, AI262449, H38876, AW269279, AW134805,AI685722, AA384219, H52328, AI701951, AAI12729, C03711, AA337888,H17067, AA299451, AA844628, AI628603, R23191, AI018039, AI767301,C04547, H15605, T99856, AA086474, AA846145, AA907827, AW050938,AA913178, T99761, AW021529, AW303950, AW269300, AI687274, AI547123,AW189104, AA496397, AA496496, AW408394, AA907899, AA496348, AA496621,AI834231, R23192, AW401626, AW059616, AF100928, AL049703, AF131759,ALF100927, AL049704, Z81364, and Z81370.

[0786] TABLE 3 Clone ID NO:Z Library Code HLD0K36 H0012 H0013 H0014H0015 H0024 H0038 H0039 H0045 H0046 H0051 H0056 H0059 H0063 H0069 H0074H0083 H0087 H0090 H0123 H0130 H0135 H0156 H0163 H0166 H0188 H0213 H0222H0250 H0261 H0264 H0265 H0266 H0271 H0294 H0295 H0309 H0316 H0333 H0341H0351 H0370 H0390 H0393 H0411 H0416 H0421 H0422 H0423 H0424 H0427 H0428H0431 H0435 H0436 H0441 H0444 H0445 H0457 H0478 H0484 H0486 H0494 H0497H0510 H0519 H0520 H0521 H0522 H0530 H0539 H0543 H0545 H0546 H0547 H0549H0551 H0556 H0574 H0575 H0576 H0580 H0581 H0584 H0585 H0587 H0594 H0595H0597 H0598 H0613 H0617 H0618 H0624 H0625 H0633 H0634 H0635 H0638 H0646H0648 H0649 H0650 H0652 H0659 H0661 H0662 H0665 H0668 H0672 H0684 H0695H0702 L1290 S0002 S0003 S0007 S0011 S0026 S0028 S0031 S0032 S0036 S0040S0045 S0046 S0049 S0050 S0052 S0114 S0116 S0126 S0132 S0142 S0144 S0150S0192 S0194 S0206 S0210 S0212 S0214 S0218 S0222 S0276 S0278 S0280 S0306S0320 S0322 S0330 S0344 S0348 S0350 S0354 S0356 S0358 S0360 S0372 S0374S0376 S0378 S0380 S0382 S0384 S0388 S0392 S0422 S0424 S0426 S0432 S0448S0450 S0460 S0472 S0474 S3012 S3014 S6022 T0002 T0003 T0006 T0039 T0042T0048 T0049 T0109 HDPBW68 H0040 H0046 H0255 H0264 H0423 H0497 H0518H0521 H0556 H0580 H0586 H0618 H0620 H0641 H0656 H0682 L1290 S0212 S0380S0460 HHEFO24 H0002 H0083 H0519 H0521 H0561 H0581 L1290 S0426 S6014HEGAL46 H0125 H0135 H0144 H0265 H0351 H0421 H0428 H0543 H0550 H0553H0592 H0617 H0618 L1290 S0002 S0418 HFOYC02 H0009 H0013 H0024 H0025H0031 H0038 H0046 H0050 H0052 H0097 H0099 H0123 H0224 H0261 H0264 H0265H0268 H0284 H0341 H0412 H0413 H0486 H0497 H0520 H0529 H0550 H0551 H0556H0595 H0599 H0616 H0637 H0638 H0658 H0688 L1290 S0002 S0010 S0026 S0027S0037 S0038 S0044 S0045 S0049 S0126 S0142 S0150 S0206 S0222 S0276 S0278S0344 S0360 S0388 S0418 S0420 S0426 S3014 T0010 T0060 T0082 T0114HDABV82 H0014 H0032 H0038 H0040 H0046 H0057 H0087 H0090 H0171 H0255H0264 H0266 H0268 H0327 H0328 H0341 H0375 H0421 H0422 H0423 H0449 H0483H0518 H0521 H0529 H0580 H0586 H0587 H0616 H0618 H0620 H0641 H0644 H0656H0657 H0672 H0682 L1290 S0003 S0045 S0176 S0212 S0214 S0222 S0356 S0380S0460 S6028 HSVAF16 H0063 H0069 H0087 H0100 H0252 H0295 H0309 H0392H0455 H0522 H0586 H0619 H0670 L1290 S0011 S0040 S0194 S0360 HSIF061H0004 H0012 H0013 H0036 H0038 H0046 H0059 H0090 H0134 H0135 H0144 H0196H0266 H0286 H0318 H0341 H0351 H0373 H0393 H0409 H0431 H0435 H0497 H0510H0521 H0522 H0529 H0542 H0543 H0551 H0556 H0561 H0590 H0592 H0638 H0644H0650 H0658 H0670 L1290 S0002 S0003 S0028 S0046 S0132 S0150 S0182 S0278S0374 T0067

[0787] TABLE 4 SEQ ID NO: Cytologic Band X or Chromosome: OMIM I.D.: 37p15-p14 107776 138079 139191 142959 153880 180104 203740 600994 601472601649 6 12 9 Xq25-q26 300037 300076 300123 301201 301845 301900 304340307150 307700 308000 308230 308240 309555 310490 312000 313350 313850

[0788] TABLE 5 Library Code Library Description H0002 Human Adult HeartH0004 Human Adult Spleen H0009 Human Fetal Brain H0012 Human FetalKidney H0013 Human 8 Week Whole Embryo H0014 Human Gall Bladder H0015Human Gall Bladder, fraction II H0024 Human Fetal Lung III H0025 HumanAdult Lymph Node H0031 Human Placenta H0032 Human Prostate H0036 HumanAdult Small Intestine H0038 Human Testes H0039 Human Pancreas TumorH0040 Human Testes Tumor H0045 Human Esophagus, Cancer H0046 HumanEndometrial Tumor H0050 Human Fetal Heart H0051 Human Hippocampus H0052Human Cerebellum H0056 Human Umbilical Vein, Endo. remake H0057 HumanFetal Spleen H0059 Human Uterine Cancer H0063 Human Thymus H0069 HumanActivated T-Cells H0074 Human Platelets H0083 HUMAN JURKAT MEMBRANEBOUND POLYSOMES H0087 Human Thymus H0090 Human T-Cell Lymphoma H0097Human Adult Heart, subtracted H0099 Human Lung Cancer, subtracted H0100Human Whole Six Week Old Embryo H0123 Human Fetal Dura Mater H0125 Cemcells cyclohexamide treated H0130 LNCAP untreated H0134 Raji Cells,cyclohexamide treated H0135 Human Synovial Sarcoma H0144 Nine Week OldEarly Stage Human H0156 Human Adrenal Gland Tumor H0163 Human SynoviumH0166 Human Prostate Cancer, Stage B2 fraction H0171 12 Week Old EarlyStage Human, II H0188 Human Normal Breast H0196 Human Cardiomyopathy,subtracted H0213 Human Pituitary, subtracted H0222 Activated T-Cells, 8hrs, subtracted H0224 Activated T-Cells, 12 hrs, subtracted H0250 HumanActivated Monocytes H0252 Human Osteosarcoma H0255 breast lymph nodeCDNA library H0261 H. cerebellum, Enzyme subtracted H0264 human tonsilsH0265 Activated T-Cell (12 hs)/Thiouridine labelledEco H0266 HumanMicrovascular Endothelial Cells, fract. A H0268 Human Umbilical VeinEndothelial Cells, fract. A H0271 Human Neutrophil, Activated H0284Human OB MG63 control fraction I H0286 Human OB MG63 treated (10 nM E2)fraction I H0294 Amniotic Cells - TNF induced H0295 Amniotic Cells -Primary Culture H0309 Human Chronic Synovitis H0316 HUMAN STOMACH H0318HUMAN B CELL LYMPHOMA H0327 human corpus colosum H0328 human ovariancancer H0333 Hemangiopericytoma H0341 Bone Marrow Cell Line (RS4,11)H0351 Glioblastoma H0370 H. Lymph node breast Cancer H0373 Human HeartH0375 Human Lung H0390 Human Amygdala Depression, re-excision H0392 H.Meningima, M1 H0393 Fetal Liver, subtraction II H0409 H. StriatumDepression, subtracted H0411 H Female Bladder, Adult H0412 Humanumbilical vein endothelial cells, IL- 4 induced H0413 Human UmbilicalVein Endothelial Cells, uninduced H0416 Human Neutrophils, Activated,re-excision H0421 Human Bone Marrow, re-excision H0422 T-Cell PHA 16 hrsH0423 T-Cell PHA 24 hrs H0424 Human Pituitary, subt IX H0427 HumanAdipose H0428 Human Ovary H0431 H. Kidney Medulla, re-excision H0435Ovarian Tumor 10-3-95 H0436 Resting T-Cell Library, II H0441 H. KidneyCortex, subtracted H0444 Spleen metastic melanoma H0445 Spleen, Chroniclymphocytic leukemia H0449 CD34 + cell, I H0455 H. Striatum Depression,subt H0457 Human Eosinophils H0478 Salivary Gland, Lib 2 H0483 BreastCancer cell line, MDA 36 H0484 Breast Cancer Cell line, angiogenic H0486Hodgkin's Lymphoma II H0494 Keratinocyte H0497 HEL cell line H0510 HumanLiver, normal H0518 pBMC stimulated w/poly I/C H0519 NTERA2, controlH0520 NTERA2 + retinoic acid, 14 days H0521 Primary Dendritic Cells, lib1 H0522 Primary Dendritic cells, frac 2 H0529 Myoloid Progenitor CellLine H0530 Human Dermal Endothelial Cells, untreated H0539 PancreasIslet Cell Tumor H0542 T Cell helper I H0543 T cell helper II H0545Human endometrial stromal cells-treated with progesterone H0546 Humanendometrial stromal cells-treated with estradiol H0547 NTERA2teratocarcinoma cell line + retinoic acid (14 days) H0549 H.Epididiymus, caput & corpus H0550 H. Epididiymus, cauda H0551 HumanThymus Stromal Cells H0553 Human Placenta H0556 Activated T-cell (12h)/Thiouridine-re- excision H0561 L428 H0574 Hepatocellular Tumor,re-excision H0575 Human Adult Pulmonary, re-excision H0576 RestingT-Cell, re-excision H0580 Dendritic cells, pooled H0581 Human BoneMarrow, treated H0584 Activated T-cells, 24 hrs, re-excision H0585Activated T-Cells, 12 hrs, re-excision H0586 Healing groin wound, 6.5hours post incision H0587 Healing groin wound, 7.5 hours post incisionH0590 Human adult small intestine, re-excision H0592 Healing groinwound - zero hr post-incision (control) H0594 Human Lung Cancer,re-excision H0595 Stomach cancer (human), re-excision H0597 Human Colon,re-excision H0598 Human Stomach, re-excision H0599 Human Adult Heart,re-excision H0613 H.Leukocytes, normalized cot 5B H0616 Human Testes,Reexcision H0617 Human Primary Breast Cancer Reexcision H0618 HumanAdult Testes, Large Inserts, Reexcision H0619 Fetal Heart H0620 HumanFetal Kidney, Reexcision H0624 12 Week Early Stage Human II, ReexcisionH0625 Ku 812F Basophils Line H0633 Lung Carcinoma A549 TNFalphaactivated H0634 Human Testes Tumor, re-excision H0635 Human ActivatedT-Cells, re-excision H0637 Dendritic Cells From CD34 Cells H0638 CD40activated monocyte dendridic cells H0641 LPS activated derived dendriticcells H0644 Human Placenta (re-excision) H0646 Lung, Cancer (4005313A3): Invasive Poorly Differentiated Lung Adenocarcinoma, H0648 Ovary,Cancer: (4004562 B6) Papillary Serous Cystic Neoplasm, Low Malignant PotH0649 Lung, Normal: (4005313 B1) H0650 B-Cells H0652 Lung, Normal:(4005313 B1) H0656 B-cells (unstimulated) H0657 B-cells (stimulated)H0658 Ovary, Cancer (9809C332): Poorly differentiated adenocarcinomaH0659 Ovary, Cancer (15395A1F): Grade II Papillary Carcinoma H0661Breast, Cancer: (4004943 A5) H0662 Breast, Normal: (4005522B2) H0665Stromal cells 3.88 H0668 stromal cell clone 2.5 H0670 Ovary, Cancer(4004650 A3): Well- Differentiated Micropapillary Serous Carcinoma H0672Ovary, Cancer: (4004576 A8) H0682 Ovarian cancer, Serous PapillaryAdenocarcinoma H0684 Ovarian cancer, Serous Papillary AdenocarcinomaH0688 Human Ovarian Cancer (#9807G017) H0695 mononucleocytes frompatient H0702 NK15 (IL2 treated for 48 hours) L1290 Stratagene HeLa cells3 937216 S0002 Monocyte activated S0003 Human Osteoclastoma S0007 EarlyStage Human Brain S0010 Human Amygdala S0011 STROMAL-OSTEOCLASTOMA S0026Stromal cell TF274 S0027 Smooth muscle, serum treated S0028 Smoothmuscle, control S0031 Spinal cord S0032 Smooth muscle-ILb induced S0036Human Substantia Nigra S0037 Smooth muscle, IL1b induced S0038 HumanWhole Brain #2 - Oligo dT > 1.5 Kb S0040 Adipocytes S0044 Prostate BPHS0045 Endothelial cells-control S0046 Endothelial-induced S0049 HumanBrain, Striatum S0050 Human Frontal Cortex, Schizophrenia S0052neutrophils control S0114 Anergic T-cell S0116 Bone marrow S0126Osteoblasts S0132 Epithelial-TNFa and INF induced S0142 Macrophage-oxLDLS0144 Macrophage (GM-CSF treated) S0150 LNCAP prostate cell line S0176Prostate, normal, subtraction I S0182 Human B Cell 8866 S0192 SynovialFibroblasts (control) S0194 Synovial hypoxia S0206 Smooth Muscle-HASTEnormalized S0210 Messangial cell, frac 2 S0212 Bone Marrow Stromal Cell,untreated S0214 Human Osteoclastoma, re-excision S0218 Apoptotic T-cell,re-excision S0222 H. Frontal cortex, epileptic, re-excision S0276Synovial hypoxia-RSF subtracted S0278 H Macrophage (GM-CSF treated), re-excision S0280 Human Adipose Tissue, re-excision S0306 Larynx normal #10261-273 S0320 Human Larynx S0322 Siebben Polyposis S0330 Palate normalS0344 Macrophage-oxLDL, re-excision S0348 Cheek Carcinoma S0350 PharynxCarcinoma S0354 Colon Normal II S0356 Colon Carcinoma S0358 Colon NormalIII S0360 Colon Tumor II S0372 Larynx carcinoma III S0374 Normal colonS0376 Colon Tumor S0378 Pancreas normal PCA4 No S0380 Pancreas TumorPCA4 Tu S0382 Larynx carcinoma IV S0384 Tongue carcinoma S0388 HumanHypothalamus, schizophrenia, re- excision S0392 Salivary Gland S0418CHME Cell Line, treated 5 hrs S0420 CHME Cell Line, untreated S0422 Mo7eCell Line GM-CSF treated (1 ng/ml) S0424 TF-1 Cell Line GM-CSF TreatedS0426 Monocyte activated, re-excision S0432 Sinus piriformis TumourS0448 Larynx Normal S0450 Larynx Tumour S0460 Thyroid Tumour S0472 LungMesothelium S0474 Human blood platelets S3012 Smooth Muscle SerumTreated, Norm S3014 Smooth muscle, serum induced, re-exc S6014 H.hypothalamus, frac A S6022 H. Adipose Tissue S6028 Human ManicDepression Tissue T0002 Activated T-cells T0003 Human Fetal Lung T0006Human Pineal Gland T0010 Human Infant Brain T0039 HSA 172 Cells T0042Jurkat T-Cell, S phase T0048 Human Aortic Endothelium T0049 Aortaendothelial cells + TNF-a T0060 Human White Adipose T0067 Human ThyroidT0082 Human Adult Retina T0109 Human (HCC) cell line liver (mouse)metastasis, remake T0114 Human (Caco-2) cell line, adenocarcinoma,colon, remake

[0789] TABLE 6 OMIM ID OMIM Description 107776 Colton blood group,110450 (3) 138079 Hyperinsulinism, familial, 602485 (3) MODY, type 2,125851 (3) 139191 Growth hormone deficient dwarfism (3) 142959Hand-foot-uterus syndrome, 140000 (3) 153880 Macular dystrophy, dominantcystoid (2) 180104 Retinitis pigmentosa-9 (2) 203740 Alpha-ketoglutaratedehydrogenase deficiency (1) 300037 Simpson dysmorphia syndrome, 312870(3) 300076 Wood neuroimmunologic syndrome (2) 300123 Mental retardationwith isolated growth hormone deficiency (2) 301201 Amelogenesisimperfecta-3, hypoplastic type (2) (?) 301845 Bazex syndrome (2) 301900Borjeson-Forssman-Lehmann syndrome (2) 304340 Mental retardation,X-linked, syndromic-5, with Dandy-Walker malformation, basal gangliadisease, and seizures (2) 307150 Hypertrichosis, congenital generalized(2) 307700 Hypoparathyroidism, X-linked (2) 308000 HPRT-related gout (3)Lesch-Nyhan syndrome (3) 308230 Immunodeficiency, X-linked, with hyper-IgM (3) 308240 Lymphoproliferative syndrome, X-linked (2) 309555Gustavson syndrome (2) 310490 Cowchock syndrome (2) 312000Panhypopituitarism, X-linked (2) 313350 Split hand/foot malformation,type 2 (2) 313850 Thoracoabdominal syndrome (2) 600994 Deafness,autosomal dominant 5 (2) 601472 Charcot-Marie-Tooth neuropathy-2D (2)601649 Blepharophimosis, epicanthus inversus, and ptosis, type 2 (2)

[0790] Having generally described the invention, the same will be morereadily understood by reference to the following examples, which areprovided by way of illustration and are not intended as limiting.

EXAMPLES Example 1

[0791] Isolation of a Selected cDNA Clone from the Deposited Sample

[0792] Each cDNA clone in a cited ATCC deposit is contained in a plasmidvector. Table 1 identifies the vectors used to construct the cDNAlibrary from which each clone was isolated. In many cases, the vectorused to construct the library is a phage vector from which a plasmid hasbeen excised. The table immediately below correlates the related plasmidfor each phage vector used in constructing the cDNA library. Forexample, where a particular clone is identified in Table 1 as beingisolated in the vector “Lambda Zap,” the corresponding deposited cloneis in “pBluescript.” Vector Used to Construct Library CorrespondingDeposited Plasmid Lambda Zap pBluescript (pBS) Uni-Zap XR pBluescript(pBS) Zap Express pBK lafmid BA plafmid BA pSport1 pSport1 pCMVSport 2.0pCMVSport 2.0 pCMVSport 3.0 pCMVSport 3.0 pCR ®2.1 pCR ®2.1

[0793] Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636),Uni-Zap XR (U.S. Pat. Nos. 5,128, 256 and 5,286,636), Zap Express (U.S.Pat. Nos. 5,128,256 and 5,286,636), pBluescript (pBS) (Short et al.,Nucleic Acids Res., 16:7583-7600 (1988); Alting-Mees et al., NucleicAcids Res., 17:9494 (1989)) and pBK (Alting-Mees et al., Strategies,5:58-61 (1992)) are commercially available from Stratagene CloningSystems, Inc., 11011 N. Torrey Pines Road, La Jolla, Calif., 92037. pBScontains an ampicillin resistance gene and pBK contains a neomycinresistance gene. Both can be transformed into E. coli strain XL-1 Blue,also available from Stratagene. pBS comes in 4 forms SK+, SK−, KS+ andKS. The S and K refers to the orientation of the polylinker to the T7and T3 primer sequences which flank the polylinker region (“S” is forSacI and “K” is for KpnI which are the first sites on each respectiveend of the linker). “+” or “−” refer to the orientation of the f1 originof replication (“ori”), such that in one orientation, single strandedrescue initiated from the f1 ori generates sense strand DNA and in theother, antisense.

[0794] Vectors pSport1, pCMVSport 2.0 and pCMVSport 3.0, were obtainedfrom Life Technologies, Inc., P. O. Box 6009, Gaithersburg, Md. 20897.All Sport vectors contain an ampicillin resistance gene and may betransformed into E. coli strain DH10B, also available from LifeTechnologies. (See, for instance, Gruber, C. E., et al., Focus 15:59(1993).) Vector lafmid BA (Bento Soares, Columbia University, NY)contains an ampicillin resistance gene and can be transformed into E.coli strain XL-1 Blue. Vector pCR®2.1, which is available fromInvitrogen, 1600 Faraday Avenue, Carlsbad, Calif. 92008, contains anampicillin resistance gene and may be transformed into E. coli strainDH10B, available from Life Technologies. (See, for instance, Clark, Nuc.Acids Res., 16:9677-9686 (1988) and Mead et al., Bio/Technology, 9(1991).) Preferably, a polynucleotide of the present invention does notcomprise the phage vector sequences identified for the particular clonein Table 1, as well as the corresponding plasmid vector sequencesdesignated above.

[0795] The deposited material in the sample assigned the ATCC DepositNumber cited in Table 1 for any given cDNA clone also may contain one ormore additional plasmids, each comprising a cDNA clone different fromthat given clone. Thus, deposits sharing the same ATCC Deposit Numbercontain at least a plasmid for each cDNA clone identified in Table 1.Typically, each ATCC deposit sample cited in Table 1 comprises a mixtureof approximately equal amounts (by weight) of about 50 plasmid DNAs,each containing a different cDNA clone; but such a deposit sample mayinclude plasmids for more or less than 50 cDNA clones, up to about 500cDNA clones.

[0796] Two approaches can be used to isolate a particular clone from thedeposited sample of plasmid DNAs cited for that clone in Table 1. First,a plasmid is directly isolated by screening the clones using apolynucleotide probe corresponding to SEQ ID NO:X.

[0797] Particularly, a specific polynucleotide with 30-40 nucleotides issynthesized using an Applied Biosystems DNA synthesizer according to thesequence reported. The oligonucleotide is labeled, for instance, with³²P-γ-ATP using T4 polynucleotide kinase and purified according toroutine methods. (E.g., Maniatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982).) The plasmidmixture is transformed into a suitable host, as indicated above (such asXL-1 Blue (Stratagene)) using techniques known to those of skill in theart, such as those provided by the vector supplier or in relatedpublications or patents cited above. The transformants are plated on1.5% agar plates (containing the appropriate selection agent, e.g.,ampicillin) to a density of about 150 transformants (colonies) perplate. These plates are screened using Nylon membranes according toroutine methods for bacterial colony screening (e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Edit., (1989), Cold SpringHarbor Laboratory Press, pages 1.93 to 1.104), or other techniques knownto those of skill in the art.

[0798] Alternatively, two primers of 17-20 nucleotides derived from bothends of the SEQ ID NO:X (i.e., within the region of SEQ ID NO:X boundedby the 5′ NT and the 3′ NT of the clone defined in Table 1) aresynthesized and used to amplify the desired cDNA using the depositedcDNA plasmid as a template. The polymerase chain reaction is carried outunder routine conditions, for instance, in 25 μl of reaction mixturewith 0.5 ug of the above cDNA template. A convenient reaction mixture is1.5-5 mM MgCl₂, 0.01% (w/v) gelatin, 20 μM each of dATP, dCTP, dGTP,dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase. Thirtyfive cycles of PCR (denaturation at 94° C. for 1 min; annealing at 55°C. for 1 min; elongation at 72° C. for 1 min) are performed with aPerkin-Elmer Cetus automated thermal cycler. The amplified product isanalyzed by agarose gel electrophoresis and the DNA band with expectedmolecular weight is excised and purified. The PCR product is verified tobe the selected sequence by subcloning and sequencing the DNA product.

[0799] Several methods are available for the identification of the 5′ or3′ non-coding portions of a gene which may not be present in thedeposited clone. These methods include but are not limited to, filterprobing, clone enrichment using specific probes, and protocols similaror identical to 5′ and 3′ “RACE” protocols which are well known in theart. For instance, a method similar to 5′ RACE is available forgenerating the missing 5′ end of a desired full-length transcript.(Fromont-Racine et al., Nucleic Acids Res., 21(7):1683-1684 (1993).)

[0800] Briefly, a specific RNA oligonucleotide is ligated to the 5′ endsof a population of RNA presumably containing full-length gene RNAtranscripts. A primer set containing a primer specific to the ligatedRNA oligonucleotide and a primer specific to a known sequence of thegene of interest is used to PCR amplify the 5′ portion of the desiredfull-length gene. This amplified product may then be sequenced and usedto generate the full length gene.

[0801] This above method starts with total RNA isolated from the desiredsource, although poly-A+ RNA can be used. The RNA preparation can thenbe treated with phosphatase if necessary to eliminate 5′ phosphategroups on degraded or damaged RNA which may interfere with the later RNAligase step. The phosphatase should then be inactivated and the RNAtreated with tobacco acid pyrophosphatase in order to remove the capstructure present at the 5′ ends of messenger RNAs. This reaction leavesa 5′ phosphate group at the 5′ end of the cap cleaved RNA which can thenbe ligated to an RNA oligonucleotide using T4 RNA ligase.

[0802] This modified RNA preparation is used as a template for firststrand cDNA synthesis using a gene specific oligonucleotide. The firststrand synthesis reaction is used as a template for PCR amplification ofthe desired 5′ end using a primer specific to the ligated RNAoligonucleotide and a primer specific to the known sequence of the geneof interest. The resultant product is then sequenced and analyzed toconfirm that the 5′ end sequence belongs to the desired gene.

Example 2

[0803] Isolation of Genomic Clones Corresponding to a Polynucleotide

[0804] A human genomic P1 library (Genomic Systems, Inc.) is screened byPCR using primers selected for the cDNA sequence corresponding to SEQ IDNO:X., according to the method described in Example 1. (See also,Sambrook.)

Example 3

[0805] Tissue Distribution of Polypeptide

[0806] Tissue distribution of mRNA expression of polynucleotides of thepresent invention is determined using protocols for Northern blotanalysis, described by, among others, Sambrook et al. For example, acDNA probe produced by the method described in Example 1 is labeled withp³² using the rediprime™ DNA labeling system (Amersham Life Science),according to manufacturer's instructions. After labeling, the probe ispurified using CHROMA SPIN-100™ column (Clontech Laboratories, Inc.),according to manufacturer's protocol number PT1200-1. The purifiedlabeled probe is then used to examine various human tissues for mRNAexpression.

[0807] Multiple Tissue Northern (MTN) blots containing various humantissues (H) or human immune system tissues (IM) (Clontech) are examinedwith the labeled probe using ExpressHyb™ hybridization solution(Clontech) according to manufacturer's protocol number PT1190-1.Following hybridization and washing, the blots are mounted and exposedto film at −70° C. overnight, and the films developed according tostandard procedures.

Example 4

[0808] Chromosomal Mapping of the Polynucleotides

[0809] An oligonucleotide primer set is designed according to thesequence at the 5′ end of SEQ ID NO:X. This primer preferably spansabout 100 nucleotides. This primer set is then used in a polymerasechain reaction under the following set of conditions: 30 seconds, 95°C.; 1 minute, 56° C.; 1 minute, 70° C. This cycle is repeated 32 timesfollowed by one 5 minute cycle at 70° C. Human, mouse, and hamster DNAis used as template in addition to a somatic cell hybrid panelcontaining individual chromosomes or chromosome fragments (Bios, Inc).The reactions is analyzed on either 8% polyacrylamide gels or 3.5%agarose gels. Chromosome mapping is determined by the presence of anapproximately 100 bp PCR fragment in the particular somatic cell hybrid.

Example 5

[0810] Bacterial Expression of a Polypeptide

[0811] A polynucleotide encoding a polypeptide of the present inventionis amplified using PCR oligonucleotide primers corresponding to the 5′and 3′ ends of the DNA sequence, as outlined in Example 1, to synthesizeinsertion fragments. The primers used to amplify the cDNA insert shouldpreferably contain restriction sites, such as BamHI and XbaI andinitiation/stop codons, if necessary, to clone the amplified productinto the expression vector. For example, BamHI and XbaI correspond tothe restriction enzyme sites on the bacterial expression vector pQE-9.(Qiagen, Inc., Chatsworth, Calif.). This plasmid vector encodesantibiotic resistance (Amp^(r)), a bacterial origin of replication(ori), an IPTG-regulatable promoter/operator (P/O), a ribosome bindingsite (RBS), a 6-histidine tag (6-His), and restriction enzyme cloningsites.

[0812] The pQE-9 vector is digested with BamHI and XbaI and theamplified fragment is ligated into the pQE-9 vector maintaining thereading frame initiated at the bacterial RBS. The ligation mixture isthen used to transform the E. coli strain M15/rep4 (Qiagen, Inc.) whichcontains multiple copies of the plasmid pREP4, which expresses the lacIrepressor and also confers kanamycin resistance (Kan^(r)). Transformantsare identified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies are selected. Plasmid DNA isisolated and confirmed by restriction analysis.

[0813] Clones containing the desired constructs are grown overnight(O/N) in liquid culture in LB media supplemented with both Amp (100ug/ml) and Kan (25 ug/ml). The ON culture is used to inoculate a largeculture at a ratio of 1:100 to 1:250. The cells are grown to an opticaldensity 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG(Isopropyl-B-D-thiogalacto pyranoside) is then added to a finalconcentration of 1 mM. IPTG induces by inactivating the lacI repressor,clearing the P/O leading to increased gene expression.

[0814] Cells are grown for an extra 3 to 4 hours. Cells are thenharvested by centrifugation (20 mins at 6000×g). The cell pellet issolubilized in the chaotropic agent 6 Molar Guanidine HCl by stirringfor 3-4 hours at 4° C. The cell debris is removed by centrifugation, andthe supernatant containing the polypeptide is loaded onto anickel-nitrilo-tri-acetic acid (“Ni—NTA”) affinity resin column(available from QIAGEN, Inc., supra). Proteins with a 6×His tag bind tothe Ni—NTA resin with high affinity and can be purified in a simpleone-step procedure (for details see: The QIAexpressionist (1995) QIAGEN,Inc., supra).

[0815] Briefly, the supernatant is loaded onto the column in 6 Mguanidine-HCl, pH 8, the column is first washed with 10 volumes of 6 Mguanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH6, and finally the polypeptide is eluted with 6 M guanidine-HCl, pH 5.

[0816] The purified protein is then renatured by dialyzing it againstphosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus200 mM NaCl. Alternatively, the protein can be successfully refoldedwhile immobilized on the Ni—NTA column. The recommended conditions areas follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl,20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. Therenaturation should be performed over a period of 1.5 hours or more.After renaturation the proteins are eluted by the addition of 250 mMimmidazole. Immidazole is removed by a final dialyzing step against PBSor 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purifiedprotein is stored at 4° C. or frozen at −80° C.

[0817] In addition to the above expression vector, the present inventionfurther includes an expression vector comprising phage operator andpromoter elements operatively linked to a polynucleotide of the presentinvention, called pHE4a. (ATCC Accession Number 209645, deposited onFeb. 25, 1998.) This vector contains: 1) a neomycinphosphotransferasegene as a selection marker, 2) an E. coli origin of replication, 3) a T5phage promoter sequence, 4) two lac operator sequences, 5) aShine-Delgarno sequence, and 6) the lactose operon repressor gene(lacIq). The origin of replication (oriC) is derived from pUC19 (LTI,Gaithersburg, Md.). The promoter sequence and operator sequences aremade synthetically.

[0818] DNA can be inserted into the pHEa by restricting the vector withNdeI and XbaI, BamHI, XhoI, or Asp718, running the restricted product ona gel, and isolating the larger fragment (the stuffer fragment should beabout 310 base pairs). The DNA insert is generated according to the PCRprotocol described in Example 1, using PCR primers having restrictionsites for NdeI (5′ primer) and XbaI, BamHI, XhoI, or Asp718 (3′ primer).The PCR insert is gel purified and restricted with compatible enzymes.The insert and vector are ligated according to standard protocols.

[0819] The engineered vector could easily be substituted in the aboveprotocol to express protein in a bacterial system.

Example 6

[0820] Purification of a Polypeptide from an Inclusion Body

[0821] The following alternative method can be used to purify apolypeptide expressed in E coli when it is present in the form ofinclusion bodies. Unless otherwise specified, all of the following stepsare conducted at 4-10° C.

[0822] Upon completion of the production phase of the E. colifermentation, the cell culture is cooled to 4-10° C. and the cellsharvested by continuous centrifugation at 15,000 rpm (Heraeus Sepatech).On the basis of the expected yield of protein per unit weight of cellpaste and the amount of purified protein required, an appropriate amountof cell paste, by weight, is suspended in a buffer solution containing100 mM Tris, 50 mM EDTA, pH 7.4. The cells are dispersed to ahomogeneous suspension using a high shear mixer.

[0823] The cells are then lysed by passing the solution through amicrofluidizer (Microfuidics, Corp. or APV Gaulin, Inc.) twice at4000-6000 psi. The homogenate is then mixed with NaCl solution to afinal concentration of 0.5 M NaCl, followed by centrifugation at 7000×gfor 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mMTris, 50 mM EDTA, pH 7.4.

[0824] The resulting washed inclusion bodies are solubilized with 1.5 Mguanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×gcentrifugation for 15 min., the pellet is discarded and the polypeptidecontaining supernatant is incubated at 4° C. overnight to allow furtherGuHCl extraction.

[0825] Following high speed centrifugation (30,000×g) to removeinsoluble particles, the GuHCl solubilized protein is refolded byquickly mixing the GuHCl extract with 20 volumes of buffer containing 50mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. Therefolded diluted protein solution is kept at 4° C. without mixing for 12hours prior to further purification steps.

[0826] To clarify the refolded polypeptide solution, a previouslyprepared tangential filtration unit equipped with 0.16 μm membranefilter with appropriate surface area (e.g., Filtron), equilibrated with40 mM sodium acetate, pH 6.0 is employed. The filtered sample is loadedonto a cation exchange resin (e.g., Poros HS-50, Perseptive Biosystems).The column is washed with 40 mM sodium acetate, pH 6.0 and eluted with250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in astepwise manner. The absorbance at 280 nm of the effluent iscontinuously monitored. Fractions are collected and further analyzed bySDS-PAGE.

[0827] Fractions containing the polypeptide are then pooled and mixedwith 4 volumes of water. The diluted sample is then loaded onto apreviously prepared set of tandem columns of strong anion (Poros HQ-50,Perseptive Biosystems) and weak anion (Poros CM-20, PerseptiveBiosystems) exchange resins. The columns are equilibrated with 40 mMsodium acetate, pH 6.0. Both columns are washed with 40 mM sodiumacetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodiumacetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractionsare collected under constant A₂₈₀ monitoring of the effluent. Fractionscontaining the polypeptide (determined, for instance, by 16% SDS-PAGE)are then pooled.

[0828] The resultant polypeptide should exhibit greater than 95% purityafter the above refolding and purification steps. No major contaminantbands should be observed from Comunassie blue stained 16% SDS-PAGE gelwhen 5 μg of purified protein is loaded. The purified protein can alsobe tested for endotoxin/LPS contamination, and typically the LPS contentis less than 0.1 ng/ml according to LAL assays.

Example 7

[0829] Cloning and Expression of a Polypeptide in a BaculovirusExpression System

[0830] In this example, the plasmid shuttle vector pA2 is used to inserta polynucleotide into a baculovirus to express a polypeptide. Thisexpression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed byconvenient restriction sites such as BamHI, Xba I and Asp718. Thepolyadenylation site of the simian virus 40 (“SV40”) is used forefficient polyadenylation. For easy selection of recombinant virus, theplasmid contains the beta-galactosidase gene from E. coli under controlof a weak Drosophila promoter in the same orientation, followed by thepolyadenylation signal of the polyhedrin gene. The inserted genes areflanked on both sides by viral sequences for cell-mediated homologousrecombination with wild-type viral DNA to generate a viable virus thatexpress the cloned polynucleotide.

[0831] Many other baculovirus vectors can be used in place of the vectorabove, such as pAc373, pVL941, and pAcIM1, as one skilled in the artwould readily appreciate, as long as the construct providesappropriately located signals for transcription, translation, secretionand the like, including a signal peptide and an in-frame AUG asrequired. Such vectors are described, for instance, in Luckow et al.,Virology 170:31-39 (1989).

[0832] Specifically, the cDNA sequence contained in the deposited cloneis amplified using the PCR protocol described in Example 1 using primerswith appropriate restriction sites and initiation/stop codons. If thenaturally occurring signal sequence is used to produce the secretedprotein, the pA2 vector does not need a second signal peptide.Alternatively, the vector can be modified (pA2 GP) to include abaculovirus leader sequence, using the standard methods described inSummers et al., “A Manual of Methods for Baculovirus Vectors and InsectCell Culture Procedures,” Texas Agricultural Experimental StationBulletin NO: 1555 (1987).

[0833] The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with appropriate restrictionenzymes and again purified on a 1% agarose gel.

[0834] The plasmid is digested with the corresponding restrictionenzymes and optionally, can be dephosphorylated using calf intestinalphosphatase, using routine procedures known in the art. The DNA is thenisolated from a 1% agarose gel using a commercially available kit(“Geneclean” BIO 101 Inc., La Jolla, Calif.).

[0835] The fragment and the dephosphorylated plasmid are ligatedtogether with T4 DNA ligase. E. coli HB101 or other suitable E. colihosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.)cells are transformed with the ligation mixture and spread on cultureplates. Bacteria containing the plasmid are identified by digesting DNAfrom individual colonies and analyzing the digestion product by gelelectrophoresis. The sequence of the cloned fragment is confirmed by DNAsequencing.

[0836] Five μg of a plasmid containing the polynucleotide isco-transfected with 1.0 μg of a commercially available linearizedbaculovirus DNA (“BaculoGold™ baculovirus DNA”, Pharmingen, San Diego,Calif.), using the lipofection method described by Felgner et al., Proc.Natl. Acad. Sci. USA 84:7413-7417 (1987). One μg of BaculoGold™ virusDNA and 5 μg of the plasmid are mixed in a sterile well of a microtiterplate containing 50 μl of serum-free Grace's medium (Life TechnologiesInc., Gaithersburg, Md.). Afterwards, 10 μl Lipofectin plus 90 μlGrace's medium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is then incubated for 5hours at 27° C. The transfection solution is then removed from the plateand 1 ml of Grace's insect medium supplemented with 10% fetal calf serumis added. Cultivation is then continued at 27° C. for four days.

[0837] After four days the supernatant is collected and a plaque assayis performed, as described by Summers and Smith, supra. An agarose gelwith “Blue Gal” (Life Technologies Inc., Gaithersburg) is used to alloweasy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay”of this type can also be found in the user's guide for insect cellculture and baculovirology distributed by Life Technologies Inc.,Gaithersburg, page 9-10.) After appropriate incubation, blue stainedplaques are picked with the tip of a micropipettor (e.g., Eppendorf).The agar containing the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 μl of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4° C.

[0838] To verify the expression of the polypeptide, Sf9 cells are grownin Grace's medium supplemented with 10% heat-inactivated FBS. The cellsare infected with the recombinant baculovirus containing thepolynucleotide at a multiplicity of infection (“MOI”) of about 2. Ifradiolabeled proteins are desired, 6 hours later the medium is removedand is replaced with SF900 II medium minus methionine and cysteine(available from Life Technologies Inc., Rockville, Md.). After 42 hours,5 μCi of ³⁵S-methionine and 5 μCi ³⁵S-cysteine (available from Amersham)are added. The cells are further incubated for 16 hours and then areharvested by centrifugation. The proteins in the supernatant as well asthe intracellular proteins are analyzed by SDS-PAGE followed byautoradiography (if radiolabeled).

[0839] Microsequencing of the amino acid sequence of the amino terminusof purified protein may be used to determine the amino terminal sequenceof the produced protein.

Example 8

[0840] Expression of a Polypeptide in Mammalian Cells

[0841] The polypeptide of the present invention can be expressed in amammalian cell. A typical mammalian expression vector contains apromoter element, which mediates the initiation of transcription ofmRNA, a protein coding sequence, and signals required for thetermination of transcription and polyadenylation of the transcript.Additional elements include enhancers, Kozak sequences and interveningsequences flanked by donor and acceptor sites for RNA splicing. Highlyefficient transcription is achieved with the early and late promotersfrom SV40, the long terminal repeats (LTRs) from Retroviruses, e.g.,RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter).

[0842] Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as pSVL and pMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146), pBC12MI (ATCC 67109), pCMVSport 2.0, and pCMVSport 3.0.Mammalian host cells that could be used include, human Hela, 293, H9 andJurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quailQC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.

[0843] Alternatively, the polypeptide can be expressed in stable celllines containing the polynucleotide integrated into a chromosome. Theco-transfection with a selectable marker such as dhfr, gpt, neomycin,hygromycin allows the identification and isolation of the transfectedcells.

[0844] The transfected gene can also be amplified to express largeamounts of the encoded protein. The DHFR (dihydrofolate reductase)marker is useful in developing cell lines that carry several hundred oreven several thousand copies of the gene of interest. (See, e.g., Alt etal., J. Biol. Chem., 253:1357-1370 (1978); Hamlin et al., Biochem. etBiophys. Acta, 1097:107-143 (1990); Page et al., Biotechnology, 9:64-68(1991)). Another useful selection marker is the enzyme glutaminesynthase (GS) (Murphy et al., Biochem J., 227:277-279 (1991); Bebbingtonet al., Bio/Technology, 10:169-175 (1992). Using these markers, themammalian cells are grown in selective medium and the cells with thehighest resistance are selected. These cell lines contain the amplifiedgene(s) integrated into a chromosome. Chinese hamster ovary (CHO) andNSO cells are often used for the production of proteins.

[0845] Derivatives of the plasmid pSV2-dhfr (ATCC Accession No.: 37146),the expression vectors pC4 (ATCC Accession No.: 209646) and pC6 (ATCCAccession No.:209647) contain the strong promoter (LTR) of the RousSarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447(March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al.,Cell, 41:521-530 (1985).) Multiple cloning sites, e.g., with therestriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate thecloning of the gene of interest. The vectors also contain the 3′ intron,the polyadenylation and termination signal of the rat preproinsulingene, and the mouse DHFR gene under control of the SV40 early promoter.

[0846] Specifically, the plasmid pC6, for example, is digested withappropriate restriction enzymes and then dephosphorylated using calfintestinal phosphates by procedures known in the art. The vector is thenisolated from a 1% agarose gel.

[0847] A polynucleotide of the present invention is amplified accordingto the protocol outlined in Example 1 using primers with appropriaterestrictions sites and initiation/stop codons, if necessary. The vectorcan be modified to include a heterologous signal sequence if necessaryfor secretion. (See, e.g., WO 96/34891.)

[0848] The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with appropriate restrictionenzymes and again purified on a 1% agarose gel.

[0849] The amplified fragment is then digested with the same restrictionenzyme and purified on a 1% agarose gel. The isolated fragment and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC6 using,for instance, restriction enzyme analysis.

[0850] Chinese hamster ovary cells lacking an active DHFR gene is usedfor transfection. Five μg of the expression plasmid pC6 is cotransfectedwith 0.5 μg of the plasmid pSVneo using lipofectin (Felgner et al.,supra). The plasmid pSV2-neo contains a dominant selectable marker, theneo gene from Tn5 encoding an enzyme that confers resistance to a groupof antibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/mlG418. After about 10-14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 μM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reversed phase HPLCanalysis.

Example 9

[0851] Protein Fusions

[0852] The polypeptides of the present invention are preferably fused toother proteins. These fusion proteins can be used for a variety ofapplications. For example, fusion of the present polypeptides toHis-tag, HA-tag, protein A, IgG domains, and maltose binding proteinfacilitates purification. (See Example 5; see also EP A 394,827;Traunecker, et al., Nature, 331:84-86 (1988)) The polypeptides can alsobe fused to heterologous polypeptide sequences to facilitate secretionand intracellular trafficking (e.g., KDEL). Moreover, fusion to IgG-1,IgG-3, and albumin increases the halflife time in vivo. Nuclearlocalization signals fused to the polypeptides of the present inventioncan target the protein to a specific subcellular localization, whilecovalent heterodimer or homodimers can increase or decrease the activityof a fusion protein. Fusion proteins can also create chimeric moleculeshaving more than one function. Finally, fusion proteins can increasesolubility and/or stability of the fused protein compared to thenon-fused protein. All of the types of fusion proteins described abovecan be made by modifying the following protocol, which outlines thefusion of a polypeptide to an IgG molecule, or the protocol described inExample 5.

[0853] Briefly, the human Fc portion of the IgG molecule can be PCRamplified, using primers that span the 5′ and 3′ ends of the sequencedescribed below. These primers also should have convenient restrictionenzyme sites that will facilitate cloning into an expression vector,preferably a mammalian expression vector, and initiation/stop codons, ifnecessary.

[0854] For example, if pC4 (Accession No.: 209646) is used, the human Fcportion can be ligated into the BamHI cloning site. Note that the 3′BamHI site should be destroyed. Next, the vector containing the human Fcportion is re-restricted with BamHI, linearizing the vector, and apolynucleotide of the present invention, isolated by the PCR protocoldescribed in Example 1, is ligated into this BamHI site. Note that thepolynucleotide is cloned without a stop codon, otherwise a fusionprotein will not be produced.

[0855] If the naturally occurring signal sequence is used to produce thesecreted protein, pC4 does not need a second signal peptide.Alternatively, if the naturally occurring signal sequence is not used,the vector can be modified to include a heterologous signal sequence.(See, e.g., WO 96/34891.) Human IgG Fc region:GGGATCCGGAGCCGAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTCG (SEQID NO:1)AGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCGATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT

Example 10

[0856] Formulating a Polypeptide

[0857] The polypeptide composition will be formulated and dosed in afashion consistent with good medical practice, taking into account theclinical condition of the individual patient (especially the sideeffects of treatment with the secreted polypeptide alone), the site ofdelivery, the method of administration, the scheduling ofadministration, and other factors known to practitioners. The “effectiveamount” for purposes herein is thus determined by such considerations.

[0858] As a general proposition, the total pharmaceutically effectiveamount of polypeptide administered parenterally per dose will be in therange of about 1 μg/kg/day to 10 mg/kg/day of patient body weight,although, as noted above, this will be subject to therapeuticdiscretion. More preferably, this dose is at least 0.01 mg/kg/day, andmost preferably for humans between about 0.01 and 1 mg/kg/day for thehormone. If given continuously, the polypeptide is typicallyadministered at a dose rate of about 1 μg/kg/hour to about 50μg/kg/hour, either by 1-4 injections per day or by continuoussubcutaneous infusions, for example, using a mini-pump. An intravenousbag solution may also be employed. The length of treatment needed toobserve changes and the interval following treatment for responses tooccur appears to vary depending on the desired effect.

[0859] Pharmaceutical compositions containing the polypeptide of theinvention are administered orally, rectally, parenterally,intracistemally, intravaginally, intraperitoneally, topically (as bypowders, ointments, gels, drops or transdermal patch), bucally, or as anoral or nasal spray. “Pharmaceutically acceptable carrier” refers to anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. The term “parenteral” asused herein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrastemal, subcutaneous andintraarticular injection and infusion.

[0860] The polypeptide is also suitably administered bysustained-release systems. Suitable examples of sustained-releasecompositions include semi-permeable polymer matrices in the form ofshaped articles, e.g., films, or mirocapsules. Sustained-releasematrices include polylactides (U.S. Pat. No.: 3,773,919, EP 58,481),copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman etal., Biopolymers, 22:547-556 (1983)), poly (2-hydroxyethyl methacrylate)(Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981), and Langer,Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate (R. Langer etal.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988). Sustained-releasecompositions also include liposomally entrapped polypeptides. Liposomescontaining the secreted polypeptide are prepared by methods known perse: U.S. Pat. No. DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci.USA, 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA,77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small(about 200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol. percent cholesterol, the selected proportionbeing adjusted for the optimal secreted polypeptide therapy.

[0861] For parenteral administration, in one embodiment, the polypeptideis formulated generally by mixing it at the desired degree of purity, ina unit dosage injectable form (solution, suspension, or emulsion), witha pharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to polypeptides.

[0862] Generally, the formulations are prepared by contacting thepolypeptide uniformly and intimately with liquid carriers or finelydivided solid carriers or both. Then, if necessary, the product isshaped into the desired formulation. Preferably the carrier is aparenteral carrier, more preferably a solution that is isotonic with theblood of the recipient. Examples of such carrier vehicles include water,saline, Ringer's solution, and dextrose solution. Non-aqueous vehiclessuch as fixed oils and ethyl oleate are also useful herein, as well asliposomes.

[0863] The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

[0864] The polypeptide is typically formulated in such vehicles at aconcentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, ata pH of about 3 to 8. It will be understood that the use of certain ofthe foregoing excipients, carriers, or stabilizers will result in theformation of polypeptide salts.

[0865] Any polypeptide to be used for therapeutic administration can besterile. Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeuticpolypeptide compositions generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

[0866] Polypeptides ordinarily will be stored in unit or multi-dosecontainers, for example, sealed ampoules or vials, as an aqueoussolution or as a lyophilized formulation for reconstitution. As anexample of a lyophilized formulation, 10-ml vials are filled with 5 mlof sterile-filtered 1% (w/v) aqueous polypeptide solution, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized polypeptide using bacteriostaticWater-for-Injection.

[0867] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition, the polypeptides of the present invention may be employed inconjunction with other therapeutic compounds.

Example 11

[0868] Method of Treating Decreased Levels of the Polypeptide

[0869] It will be appreciated that conditions caused by a decrease inthe standard or normal expression level of a polypeptide in anindividual can be treated by administering the polypeptide of thepresent invention, preferably in the secreted and/or soluble form. Thus,the invention also provides a method of treatment of an individual inneed of an increased level of the polypeptide comprising administeringto such an individual a pharmaceutical composition comprising an amountof the polypeptide to increase the activity level of the polypeptide insuch an individual.

[0870] For example, a patient with decreased levels of a polypeptidereceives a daily dose 0.1-100 ug/kg of the polypeptide for sixconsecutive days. Preferably, the polypeptide is in the secreted form.The exact details of the dosing scheme, based on administration andformulation, are provided in Example 10.

Example 12

[0871] Method of Treating Increased Levels of the Polypeptide

[0872] Antisense technology is used to inhibit production of apolypeptide of the present invention. This technology is one example ofa method of decreasing levels of a polypeptide, preferably a secretedform, due to a variety of etiologies, such as cancer.

[0873] For example, a patient diagnosed with abnormally increased levelsof a polypeptide is administered intravenously antisense polynucleotidesat 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment isrepeated after a 7-day rest period if the treatment was well tolerated.The formulation of the antisense polynucleotide is provided in Example10.

Example 13

[0874] Method of Treatment Using Gene Therapy—Ex Vivo

[0875] One method of gene therapy transplants fibroblasts, which arecapable of expressing a polypeptide, onto a patient. Generally,fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin) is added. The flasks are then incubated at 37° C. forapproximately one week.

[0876] At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks.

[0877] pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flankedby the long terminal repeats of the Moloney murine sarcoma virus, isdigested with EcoRI and HindIII and subsequently treated with calfintestinal phosphatase. The linear vector is fractionated on agarose geland purified, using glass beads.

[0878] The cDNA encoding a polypeptide of the present invention can beamplified using PCR primers which correspond to the 5′ and 3′ endsequences respectively as set forth in Example 1 using primers andhaving appropriate restriction sites and initiation/stop codons, ifnecessary. Preferably, the 5′ primer contains an EcoRI site and the 3′primer includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is then used totransform bacteria HB101, which are then plated onto agar containingkanamycin for the purpose of confirming that the vector has the gene ofinterest properly inserted.

[0879] The amphotropic pA317 or GP+am12 packaging cells are grown intissue culture to confluent density in Dulbecco's Modified Eagles Medium(DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSVvector containing the gene is then added to the media and the packagingcells transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

[0880] Fresh media is added to the transduced producer cells, andsubsequently, the media is harvested from a 10 cm plate of confluentproducer cells. The spent media, containing the infectious viralparticles, is filtered through a millipore filter to remove detachedproducer cells and this media is then used to infect fibroblast cells.Media is removed from a sub-confluent plate of fibroblasts and quicklyreplaced with the media from the producer cells. This media is removedand replaced with fresh media. If the titer of virus is high, thenvirtually all fibroblasts will be infected and no selection is required.If the titer is very low, then it is necessary to use a retroviralvector that has a selectable marker, such as neo or his. Once thefibroblasts have been efficiently infected, the fibroblasts are analyzedto determine whether protein is produced.

[0881] The engineered fibroblasts are then transplanted onto the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads.

Example 14

[0882] Gene Therapy Using Endogenous Apoptosis Related Genes

[0883] Another method of gene therapy according to the present inventioninvolves operably associating the endogenous apoptosis related genesequence with a promoter via homologous recombination as described, forexample, in U.S. Pat. No.: 5,641,670, issued Jun. 24, 1997;International Publication NO: WO 96/29411, published Sep. 26, 1996;International Publication NO: WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); andZijlstra et al., Nature, 342:435-438 (1989). This method involves theactivation of a gene which is present in the target cells, but which isnot expressed in the cells, or is expressed at a lower level thandesired.

[0884] Polynucleotide constructs are made which contain a promoter andtargeting sequences, which are homologous to the 5′ non-coding sequenceof the endogenous apoptosis related gene, flanking the promoter. Thetargeting sequence will be sufficiently near the 5′ end of apoptosisrelated gene so the promoter will be operably linked to the endogenoussequence upon homologous recombination. The promoter and the targetingsequences can be amplified using PCR. Preferably, the amplified promotercontains distinct restriction enzyme sites on the 5′ and 3′ ends.Preferably, the 3′ end of the first targeting sequence contains the samerestriction enzyme site as the 5′ end of the amplified promoter and the5′ end of the second targeting sequence contains the same restrictionsite as the 3′ end of the amplified promoter.

[0885] The amplified promoter and the amplified targeting sequences aredigested with the appropriate restriction enzymes and subsequentlytreated with calf intestinal phosphatase. The digested promoter anddigested targeting sequences are added together in the presence of T4DNA ligase. The resulting mixture is maintained under conditionsappropriate for ligation of the two fragments. The construct is sizefractionated on an agarose gel then purified by phenol extraction andethanol precipitation.

[0886] In this Example, the polynucleotide constructs are administeredas naked polynucleotides via electroporation. However, thepolynucleotide constructs may also be administered withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, precipitating agents, etc. Such methods of delivery areknown in the art.

[0887] Once the cells are transfected, homologous recombination willtake place which results in the promoter being operably linked to theendogenous apoptosis related gene sequence. This results in theexpression of apoptosis related in the cell. Expression may be detectedby immunological staining, or any other method known in the art.

[0888] Fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in DMEM+10% fetal calf serum. Exponentiallygrowing or early stationary phase fibroblasts are trypsinized and rinsedfrom the plastic surface with nutrient medium. An aliquot of the cellsuspension is removed for counting, and the remaining cells aresubjected to centrifugation. The supernatant is aspirated and the pelletis resuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3,137 mM NaCl, 5 mM KCl, 0.7 mM Na₂ HPO₄, 6 mM dextrose). The cells arerecentrifuged, the supernatant aspirated, and the cells resuspended inelectroporation buffer containing 1 mg/ml acetylated bovine serumalbumin. The final cell suspension contains approximately 3×10⁶cells/ml. Electroporation should be performed immediately followingresuspension.

[0889] Plasmid DNA is prepared according to standard techniques. Forexample, to construct a plasmid for targeting to the apoptosis relatedlocus, plasmid pUC18 (MBI Fermentas, Amherst, N.Y.) is digested withHindIII. The CMV promoter is amplified by PCR with an XbaI site on the5′ end and a BamHI site on the 3′end. Two apoptosis related non-codinggene sequences are amplified via PCR: one apoptosis related non-codingsequence (apoptosis related fragment 1) is amplified with a HindIII siteat the 5′ end and an Xba site at the 3′end; the other apoptosis relatednon-coding sequence (apoptosis related fragment 2) is amplified with aBamHI site at the 5′end and a HindIII site at the 3′end. The CMVpromoter and apoptosis related fragments are digested with theappropriate enzymes (CMV promoter—XbaI and BamHI; apoptosis relatedfragment 1—XbaI; apoptosis related fragment 2—BamHI) and ligatedtogether. The resulting ligation product is digested with HindIII, andligated with the HindIII-digested pUC 18 plasmid.

[0890] Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrodegap (Bio-Rad). The final DNA concentration is generally at least 120μg/ml. 0.5 ml of the cell suspension (containing approximately 1.5.×10⁶cells) is then added to the cuvette, and the cell suspension and DNAsolutions are gently mixed. Electroporation is performed with aGene-Pulser apparatus (Bio-Rad). Capacitance and voltage are set at 960μF and 250-300 V, respectively. As voltage increases, cell survivaldecreases, but the percentage of surviving cells that stably incorporatethe introduced DNA into their genome increases dramatically. Given theseparameters, a pulse time of approximately 14-20 mSec should be observed.

[0891] Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cmdish and incubated at 37 degree C. The following day, the media isaspirated and replaced with 10 ml of fresh media and incubated for afurther 16-24 hours.

[0892] The engineered fibroblasts are then injected into the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads. The fibroblasts now produce the protein product. Thefibroblasts can then be introduced into a patient as described above.

Example 15

[0893] Method of Treatment Using Gene Therapy—In Vivo

[0894] Another aspect of the present invention is using in vivo genetherapy methods to treat disorders, diseases and conditions. The genetherapy method relates to the introduction of naked nucleic acid (DNA,RNA, and antisense DNA or RNA) apoptosis related sequences into ananimal to increase or decrease the expression of the apoptosis relatedpolypeptide. The apoptosis related polynucleotide may be operativelylinked to a promoter or any other genetic elements necessary for theexpression of the apoptosis related polypeptide by the target tissue.Such gene therapy and delivery techniques and methods are known in theart, see, for example, WO90/11092, WO98/11779; U.S. Pat. Nos.: 5693622,5705151, 5580859; Tabata et al., Cardiovasc. Res. 35(3):470-479 (1997),Chao J et al., Pharmacol. Res., 35(6):517-522 (1997), Wolff,Neuromuscul. Disord. 7(5):314-318 (1997), Schwartz et al., Gene Ther.,3(5):405-411 (1996), Tsurumi Y. et al., Circulation, 94(12):3281-3290(1996) (incorporated herein by reference).

[0895] The apoptosis related polynucleotide constructs may be deliveredby any method that delivers injectable materials to the cells of ananimal, such as, injection into the interstitial space of tissues(heart, muscle, skin, lung, liver, intestine and the like). Theapoptosis related polynucleotide constructs can be delivered in apharmaceutically acceptable liquid or aqueous carrier.

[0896] The term “naked” polynucleotide, DNA or RNA, refers to sequencesthat are free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, the apoptosis related polynucleotides may also bedelivered in liposome formulations (such as those taught in Felgner etal., Ann. NY Acad. Sci., 772:126-139 (1995) and Abdallah et al., Biol.Cell , 85(1):1-7 (1995)) which can be prepared by methods well known tothose skilled in the art.

[0897] The apoptosis related polynucleotide vector constructs used inthe gene therapy method are preferably constructs that will notintegrate into the host genome nor will they contain sequences thatallow for replication. Any strong promoter known to those skilled in theart can be used for driving the expression of DNA. Unlike other genetherapies techniques, one major advantage of introducing naked nucleicacid sequences into target cells is the transitory nature of thepolynucleotide synthesis in the cells. Studies have shown thatnon-replicating DNA sequences can be introduced into cells to provideproduction of the desired polypeptide for periods of up to six months.

[0898] The polynucleotide constructs can be delivered to theinterstitial space of tissues within the an animal, including of muscle,skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph,blood, bone, cartilage, pancreas, kidney, gall bladder, stomach,intestine, testis, ovary, uterus, rectum, nervous system, eye, gland,and connective tissue. Interstitial space of the tissues comprises theintercellular fluid, mucopolysaccharide matrix among the reticularfibers of organ tissues, elastic fibers in the walls of vessels orchambers, collagen fibers of fibrous tissues, or that same matrix withinconnective tissue ensheathing muscle cells or in the lacunae of bone. Itis similarly the space occupied by the plasma of the circulation and thelymph fluid of the lymphatic channels. Delivery to the interstitialspace of muscle tissue is preferred for the reasons discussed below.They may be conveniently delivered by injection into the tissuescomprising these cells. They are preferably delivered to and expressedin persistent, non-dividing cells which are differentiated, althoughdelivery and expression may be achieved in non-differentiated or lesscompletely differentiated cells, such as, for example, stem cells ofblood or skin fibroblasts. In vivo muscle cells are particularlycompetent in their ability to take up and express polynucleotides.

[0899] For the naked apoptosis related polynucleotide injection, aneffective dosage amount of DNA or RNA will be in the range of from about0.05 g/kg body weight to about 50 mg/kg body weight. Preferably thedosage will be from about 0.005 mg/kg to about 20 mg/kg and morepreferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as theartisan of ordinary skill will appreciate, this dosage will varyaccording to the tissue site of injection. The appropriate and effectivedosage of nucleic acid sequence can readily be determined by those ofordinary skill in the art and may depend on the condition being treatedand the route of administration. The preferred route of administrationis by the parenteral route of injection into the interstitial space oftissues. However, other parenteral routes may also be used, such as,inhalation of an aerosol formulation particularly for delivery to lungsor bronchial tissues, throat or mucous membranes of the nose. Inaddition, naked apoptosis related polynucleotide constructs can bedelivered to arteries during angioplasty by the catheter used in theprocedure.

[0900] The dose response effects of injected apoptosis relatedpolynucleotide in muscle in vivo is determined as follows. Suitableapoptosis related template DNA for production of mRNA coding forapoptosis related polypeptide is prepared in accordance with a standardrecombinant DNA methodology. The template DNA, which may be eithercircular or linear, is either used as naked DNA or complexed withliposomes. The quadriceps muscles of mice are then injected with variousamounts of the template DNA.

[0901] Five to six week old female and male Balb/C mice are anesthetizedby intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cmincision is made on the anterior thigh, and the quadriceps muscle isdirectly visualized. The apoptosis related template DNA is injected in0.1 ml of carrier in a 1 cc syringe through a 27 gauge needle over oneminute, approximately 0.5 cm from the distal insertion site of themuscle into the knee and about 0.2 cm deep. A suture is placed over theinjection site for future localization, and the skin is closed withstainless steel clips.

[0902] After an appropriate incubation time (e.g., 7 days) muscleextracts are prepared by excising the entire quadriceps. Every fifth 15um cross-section of the individual quadriceps muscles is histochemicallystained for apoptosis related protein expression. A time course forapoptosis related protein expression may be done in a similar fashionexcept that quadriceps from different mice are harvested at differenttimes. Persistence of apoptosis related DNA in muscle followinginjection may be determined by Southern blot analysis after preparingtotal cellular DNA and HIRT supernatants from injected and control mice.The results of the above experimentation in mice can be use toextrapolate proper dosages and other treatment parameters in humans andother animals using apoptosis related naked DNA.

Example 16

[0903] Production of an Antibody

[0904] a) Hybridoma Technology

[0905] The antibodies of the present invention can be prepared by avariety of methods. (See, Current Protocols, Chapter 2.) As one exampleof such methods, cells expressing apoptosis related polypeptide(s) areadministered to an animal to induce the production of sera containingpolyclonal antibodies. In a preferred method, a preparation of apoptosisrelated polypeptide(s) is prepared and purified to render itsubstantially free of natural contaminants. Such a preparation is thenintroduced into an animal in order to produce polyclonal antisera ofgreater specific activity.

[0906] Monoclonal antibodies specific for apoptosis relatedpolypeptide(s) are prepared using hybridoma technology. (Kohler et al.,Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976);Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in:Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681(1981)). In general, an animal (preferably a mouse) is immunized withapoptosis related polypeptide(s) or, more preferably, with a secretedapoptosis related polypeptide-expressing cell. Suchpolypeptide-expressing cells are cultured in any suitable tissue culturemedium, preferably in Earle's modified Eagle's medium supplemented with10% fetal bovine serum (inactivated at about 56° C.), and supplementedwith about 10 g/l of nonessential amino acids, about 1,000 U/ml ofpenicillin, and about 100 μg/ml of streptomycin.

[0907] The splenocytes of such mice are extracted and flised with asuitable myeloma cell line. Any suitable myeloma cell line may beemployed in accordance with the present invention; however, it ispreferable to employ the parent myeloma cell line (SP2O), available fromthe ATCC. After filsion, the resulting hybridoma cells are selectivelymaintained in HAT medium, and then cloned by limiting dilution asdescribed by Wands et al. (Gastroenterology 80:225-232 (1981)). Thehybridoma cells obtained through such a selection are then assayed toidentify clones which secrete antibodies capable of binding theapoptosis related polypeptide(s).

[0908] Alternatively, additional antibodies capable of binding toapoptosis related polypeptide(s) can be produced in a two-step procedureusing anti-idiotypic antibodies. Such a method makes use of the factthat antibodies are themselves antigens, and therefore, it is possibleto obtain an antibody which binds to a second antibody. In accordancewith this method, protein specific antibodies are used to immunize ananimal, preferably a mouse. The splenocytes of such an animal are thenused to produce hybridoma cells, and the hybridoma cells are screened toidentify clones which produce an antibody whose ability to bind to theapoptosis related protein-specific antibody can be blocked by apoptosisrelated polypeptide(s). Such antibodies comprise anti-idiotypicantibodies to the apoptosis related protein-specific antibody and areused to immunize an animal to induce formation of further apoptosisrelated protein-specific antibodies.

[0909] For in vivo use of antibodies in humans, an antibody is“humanized”. Such antibodies can be produced using genetic constructsderived from hybridoma cells producing the monoclonal antibodiesdescribed above. Methods for producing chimeric and humanized antibodiesare known in the art and are discussed herein. (See, for review,Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533;Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984);Neuberger et al., Nature 314:268 (1985).)

[0910] b) Isolation Of Antibody Fragments Directed Against apoptosisrelated Polypeptide(s) From A Library Of scFvs

[0911] Naturally occurring V-genes isolated from human PBLs areconstructed into a library of antibody fragments which containreactivities against apoptosis related polypeptide(s) to which the donormay or may not have been exposed (see e.g., U.S. Pat. No. 5,885,793incorporated herein by reference in its entirety).

[0912] Rescue of the Library.

[0913] A library of scFvs is constructed from the RNA of human PBLs asdescribed in PCT publication WO 92/01047. To rescue phage displayingantibody fragments, approximately 109 E. coli harboring the phagemid areused to inoculate 50 ml of 2×TY containing 1% glucose and 100 μg/ml ofampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8 with shaking. Fiveml of this culture is used to innoculate 50 ml of 2×TY-AMP-GLU, 2×108 TUof delta gene 3 helper (M13 delta gene III, see PCT publication WO92/01047) are added and the culture incubated at 37° C. for 45 minuteswithout shaking and then at 37° C. for 45 minutes with shaking. Theculture is centrifuged at 4000 r.p.m. for 10 min. and the pelletresuspended in 2 liters of 2×TY containing 100 μg/ml ampicillin and 50ug/ml kanamycin and grown overnight. Phage are prepared as described inPCT publication WO 92/01047.

[0914] M13 delta gene III is prepared as follows: M13 delta gene IIIhelper phage does not encode gene III protein, hence the phage(mid)displaying antibody fragments have a greater avidity of binding toantigen. Infectious M13 delta gene III particles are made by growing thehelper phage in cells harboring a pUC19 derivative supplying the wildtype gene III protein during phage morphogenesis. The culture isincubated for 1 hour at 37° C. without shaking and then for a furtherhour at 37° C. with shaking. Cells are spun down (IEC-Centra 8,400r.p.m. for 10 min), resuspended in 300 ml 2×TY broth containing 100 μgampicillin/ml and 25 μg kanamycin/ml (2×TY-AMP-KAN) and grown overnight,shaking at 37° C. Phage particles are purified and concentrated from theculture medium by two PEG-precipitations (Sambrook et al., 1990),resuspended in 2 ml PBS and passed through a 0.45 μm filter (MinisartNML; Sartorius) to give a final concentration of approximately 1013transducing units/ml (ampicillin-resistant clones).

[0915] Panning of the Library.

[0916] Immunotubes (Nunc) are coated overnight in PBS with 4 ml ofeither 100 μg/ml or 10 ug/ml of a polypeptide of the present invention.Tubes are blocked with 2% Marvel-PBS for 2 hours at 37° C. and thenwashed 3 times in PBS. Approximately 1013 TU of phage is applied to thetube and incubated for 30 minutes at room temperature tumbling on anover and under turntable and then left to stand for another 1.5 hours.Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS.Phage are eluted by adding 1 ml of 100 mM triethylamine and rotating 15minutes on an under and over turntable after which the solution isimmediately neutralized with 0.5 ml of 1.0M Tris-HCl, pH 7.4. Phage arethen used to infect 10 ml of mid-log E. coli TG1 by incubating elutedphage with bacteria for 30 minutes at 37° C. The E. coli are then platedon TYE plates containing 1% glucose and 100 μg/ml ampicillin. Theresulting bacterial library is then rescued with delta gene 3 helperphage as described above to prepare phage for a subsequent round ofselection. This process is then repeated for a total of 4 rounds ofaffinity purification with tube-washing increased to 20 times with PBS,0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

[0917] Characterization of Binders.

[0918] Eluted phage from the 3rd and 4th rounds of selection are used toinfect E. coli HB 2151 and soluble scFv is produced (Marks, et al.,1991) from single colonies for assay. ELISAs are performed withmicrotitre plates coated with either 10 pg/ml of the polypeptide of thepresent invention in 50 mM bicarbonate pH 9.6. Clones positive in ELISAare further characterized by PCR fingerprinting (see, e.g., PCTpublication WO 92/01047) and then by sequencing. These ELISA positiveclones may also be further characterized by techniques known in the art,such as, for example, epitope mapping, binding affinity, receptor signaltransduction, ability to block or competitively inhibit antibody/antigenbinding, and competitive agonistic or antagonistic activity.

Example 17

[0919] Identification of Binding Partners:

[0920] The CARD and its structurally related death effector domain (DED)are peptide motifs that mediate interactions betweenapoptosis-regulatory proteins (Science 281:1301 (1998); Hofmann et al.,Trends Biochem. Sci. 22:155 (1998)). To identify the binding partners ofNod1, the ability of Nod1 to associate with a panel of CARD andDED-containing proteins in 293T cells was tested. In these experiments,Nod1 is Myc- or HA-epitope-tagged and transiently expressed in 293Tcells. Overexpressed Nod1 is a cytosolic protein. Co-immunoprecipitationassays with Nod1 and tagged proteins reveal that Nod1 preferentiallyinteracts with several procaspases containing long prodomains with CARDsor DEDs including Caspase-1, Caspase-2, Caspase-4, Caspase-8 andCaspase-9, but not with short prodomains like Caspase-3 or Caspase-7(Inohara, et al., J. Biol. Chem. 273:12296 (1998); Inohara et al.,unpublished data)). In addition, Nod1 interacts with Nod1 itself, withRICK, ARC, IAP-1 and NIAP, but not with other CARD or DED-containingproteins including RAIDD, Apaf-1, Ced-4, FADD, PEA15, or DEDD. Inaddition, Nod1 does not associate with IAP1 or several TRAFs, nor withmultiple regulators of the NF-kB activation including several TRAFs,IKKa, IKKb, N1K, or A20.

Example 18

[0921] Functional Activity of Apoptosis Related Polypeptides

[0922] Modulation of Apoptosis

[0923] To determine the functional relevance of the above interactions,the ability of Nod1 to modulate the apoptosis regulatory function ofbinding proteins in 293T cells is tested (Inohara et al., J. Biol.Chem., 273:12296 (1998); McCarthy et al., J. Biol. Chem., 273:16968(1998); Thorne et al., Curr. Biol., 8:885 (1998)). Nod1 does notactivate apoptosis by itself, but significantly enhances apoptosisinduced by Caspase-9, but not that induced by Caspase-4, Caspase-8, orCLARP. Consistent with its inability to modulate Caspase-8-mediatedapoptosis, Nod1 does not affect apoptosis induced by FADD, CLARP,DR3/TRAMP and TNRF1, which induce apoptosis through Caspase-8activation. (For review, see the special issue of Science 281:1301(1998)). In addition, expression of Nod1 does not change the apoptosisregulatory function of Caspase-1, Caspase-2, RICK, ARC, IAP-1, and NIAP.These results indicate that the ability of Nod1 to regulate apoptosis ishighly specific and limited so far to that activated by Caspase-9. Theeffect of Nod1 on Caspase-9-induced apoptosis is not due to increasedexpression of Caspase-9, as there is not alteration in the levels ofthis procaspase-9 in cells transfected with Nod1 plasmids compared tocontrol plasmids. Production of enzymatically active caspases includingCaspase-9, requires proteolytic processing of the immature form of theenzyme. (For review, see the special issue of Science 281:1301 (1998)).To determine if Nod1 enhances Caspase-9 processing, 293T cells aretransiently expressed in the presence or absence of Nod1. (Total lysatefrom 293T cells transfected with 50 ng of pcDNA3-Caspase-9-Flag orpcDNA3-Caspase-8-HA in the presence of 100 ng of pcDNA3-HA orpcDNA3-Nod1-HA is prepared 24 hr post-transfection and is subject to 15%SDS polyacrylamide gel electrophoresis. Tagged Caspase-9 and Caspase-8are detected by anti-Flag and anti-HA antibodies, respectively).Expression of Nod1 includes the proteolytic activation of procaspase-9,but not that of procaspase-8. Thus, the ability of Nod1 to enhancecaspase-9 but not caspase-8-mediated apoptosis correlates with itsability to induce proteolytic processing of procaspase. By analogy withNod1, Apaf-1 binds to several caspases with long prodomains, but onlypromotes activation of procaspase-9. (Hu et al., Proc. Natl. Acad. Sci.USA 95:4386 (1998)).

[0924] Identification of Functionally Important Residues

[0925] Mutant forms of Nod1 (polypeptides comprising residues 1-648,649-953, and 126-953) are engineered to determine the regions of Nod1that are required for caspase-9 activation. Expression of mutantcontaining the CARD and NBD (residues 1-648) retains its ability toenhance Caspase-9-induce apoptosis but the mutant containing the LRRs(residues 659-953) does not, indicating that Nod1 promotes Caspase-9apoptosis through the CARD and/or NBD, as it was previously reported forApaf-1 and Ced-4. (Zou et al., Cell, 90:405 (1997); Hu et al., Proc.Natl. Acad. Sci. USA, 95:4386 (1998); and Chinnaiyan et al., Nature,388:728 (1997); Hu et al., Proc. Natl. Acad. Sci. USA, 95:4386 (1998);Srinivasula et al., Mol. Cell, 1:946 (1998); Chaudhary et al., J. Biol.Chem., 273:17708 (1998); Yang et al., Science, 281:1355 (1998)). Theconserved lysine residue of P-loop of ced-4 and Apaf-1 are critical forboth caspase activation and apoptosis enhancement (Hu et al., Proc.Natl. Acad. Sci. USA, 95:4386 (1998); and Chinnaiyan et al., Nature,388:728 (1997); Hu et al., Proc. Natl. Acad. Sci. USA, 95:4386 (1998);Srinivasula et al., Mol. Cell, 1:946 (1998); Chaudhary et al., J. Biol.Chem., 273:17708 (1998); Yang et al., Science, 281:1355 (1998)) as wellas the conserved residues in the CARD of RAIDD (Duan et al. Nature,385:86 (1997); Chou et al., Cell, 94:171 (1998)). Therefore, pointmutations are introduced in highly conserved residues of the CARD (V41Q)and in the P-loop of the NBD (K208R), whose corresponding mutationresults in loss-of-function of RAIDD, Apaf-1/Ced-4, respectively. (Hu etal., Proc. Natl. Acad. Sci. USA, 95:4386 (1998); Chinnaiyan et al.,Nature, 388:728 (1997); Hu et al., Proc. Natl. Acad. Sci. USA, 95:4386(1998); Srinivasula et al., Mol. Cell, 1:946 (1998); Chaudhary et al.,J. Biol. Chem., 273:17708 (1998); Yang et al., Science, 281:1355 (1998);Duan et al. Nature, 385:86 (1997); Chou et al., Cell, 94:171 (1998)).Both V41Q and K208R mutations of Nod1 inhibit the ability of Nod1 toenhance Caspase-9-induced apoptosis and Caspase-9 maturation. Thus, theCARD and NBD appear essential for Nod1 to activate procaspas-9 and topromote apoptosis. These results suggest that Nod1 and Apaf-1 activateprocaspase-9 by a similar mechanisms, which may involve conformationchanges and NBD oligomerization of these caspase activators to bring inclose proximity several molecules of procaspase-9 (Hu et al., Proc.Natl. Acad. Sci. USA, 95:4386 (1998); Chinnaiyan et al., Nature, 388:728(1997); Hu et al., Proc. Natl. Acad. Sci. USA, 95:4386 (1998);Srinivasula et al., Mol. Cell, 1:946 (1998); Chaudhary et al., J. Biol.Chem., 273:17708 (1998); Yang et al., Science, 281:1355 (1998)).

[0926] Identification of Regions Interacting with Binding Partners

[0927] Both Ced-4 and Apaf-1 associate with their target caspases viatheir corresponding CARDs (Zou et al., Cell, 90:405 (1997); Hu et al.,Proc. Natl. Acad. Sci. USA, 95:4386 (1998); ); Chinnaiyan et al.,Nature, 388:728 (1997); Hu et al., Proc. Natl. Acad. Sci. USA, 95:4386(1998); Srinivasula et al., Mol. Cell, 1:946 (1998); Chaudhary et al.,J. Biol. Chem., 273:17708 (1998); Yang et al., Science, 281:1355(1998)). Therefore the regions of Nod1 that are required for associationwith procaspase-9 are determined. 293T cells are transientlyco-transfected with expression plasmids producing Caspase-9 andwild-type or mutant forms of Nod1. Immunoblotting analysis of proteincomplexes reveals that residues 1-648 of Nod1 containing the CARD andNBD co-immunoprecipitates with Caspase-9 but the LRRs does not. TheK208R mutant still binds to Caspase-9, although its binding is reducedwhen compared with wild-type Nod1. Nod1 with mutation in a highlyconserved residue of the CARD (V41Q) fails to associate with Caspase-9C287S. Another mutant lacking the CARD (residues 126-953) also fails tointeract with and activate procaspase-9. Thus, the CARD is essential forNod-1 to bind and to activate procaspase-9, as well as to promoteapoptosis. These results are consistent with the analyses of theApaf-1-Caspase-9 and Ced-4-Ced-3 interactions that shows that the CARDof Apaf-1 and Ced-4 are involved in their interaction with Caspase-9 andCed-3, respectively.

[0928] These results show that Nod1 also interacts with RICK, aserine-threonine kinase with a CARD that promotes apoptosis and NF-kBactivation. (Inohara et al., J. Biol. Chem. 273:12296 (1998); McCarthyet al., J. Biol. Chem. 273:16968 (1998); Thorne et al., Curr. Biol.,8:885 (1998)). Because RICK induces NF-kB activation, it is determinedwhether Nod-1 could activate NF-kB. To test if Nod1 activates NF-kB, aNod1 expression plasmid is co-transfected with pBIIx-Luc, a luciferaseNF-kB reported plasmid or pf-Luc control plasmid lacking NF-kB bindingsites into 293T cells (Inohara et al., J. Biol. Chem. 273:12296 (1998);McCarthy et al., J. Biol. Chem. 273:16968 (1998); Thorne et al., Curr.Biol., 8:885 (1998); Saksela et al., Mol. Cell. Biol., 13:3698 (1993)).Nod1 induces activation of NF-kB in a dose-dependent manner, whereasApaf-1 does not. It is also confirmed that Nod1 induces NF-kB activationin parental 293 cells and HeLa cells. RICK alone induces NF-kBactivation as reported (Inohara et al., J. Biol. Chem., 273:12296(1998); McCarthy et al., J. Biol. Chem., 273:16968 (1998); Thorne etal., Curr. Biol., 8:885 (1998)), but co-expression of RICK and Nod1causes synergistic NF-kB activation.

[0929] Mapping of Site of Action in NF-kB Pathway

[0930] Several surface receptors and intracellular mediators usedifferent transducing molecules to activate a common set ofintracellular components that lead to degradation of I-kB and release ofcytoplasmic NF-kB such as RIP, TRAF2 and TRAF6. (Baeuerle et al., Cell,87:13 (1996); Stancovski et al., Cell, 91:299 (1997); Verma et al.,Proc. Natl. Acad. Sci. USA, 94:11758 (1997); Arch et al., Genes Dev.,12:2821 (1998); T. Maniatis, Science, 278:818 (1998)). Stimulation ofthese upstream components culminate in the activation of a common set ofsignaling molecules that include NIK and IKKs that lead to inactivationof I-kB. (Baeuerle et al., Cell, 87:13 (1996); Stancovski et al., Cell,91:299 (1997); Verma et al., Proc. Natl. Acad. Sci. USA, 94:11758(1997); Arch et al., Genes Dev., 12:2821 (1998); T. Maniatis, Science,278:818 (1998)). Mutant forms of these signaling components are used tomap the site of Nod1 action in the NF-kB activation pathway. The NF-kBactivity of Nod1 is abolished or greatly inhibited by dominant negativeforms of NIK, IKKα, IKKβ and I-kB, but not by mutant forms of TRAF2,TRAF6 or RIP which can inhibit NF-kB activation mediated by surfacereceptors. (Baeuerle et al., Cell, 87:13 (1996); Stancovski et al.,Cell, 91:299 (1997); Verma et al., Proc. Natl. Acad. Sci. USA, 94:11758(1997); Arch et al., Genes Dev., 12:2821 (1998); T. Maniatis, Science,278:818 (1998); Hsu et al., Cell, 84:299 (1996); Cao et al., Nature,383:443 (1996); Hsu et al., Immunity, 4:387 (1996); Liu et al., Cell,87:565 (1996)). To test if Nod-1 activates NF-kB independently of itsability to promote Caspase-9-induced apoptosis, a catalytically inactivemutant of caspase-9 that acts as a dominant negative is used. Pan etal., J. Biol. Chem., 273:5841 (1998); Inohara et al., J. Biol. Chem.,273:32479 (1998). Expression of mutant Caspase-9 does not inhibit theactivation of NF-kB induced by Nod1. These results suggest that Nod1acts upstream of NIK, IKKs and I-kB kB but in a different pathway, ordownstream of TRAF2, TRAF6 or RIP. Moreover, the result with dominantnegative Caspase-9 suggests that the NF-kB-inducing activity of Nod1does not require Caspase-9 activity. To determine the regions of Nod1and RICK involved in the interaction, 293T cells are transientlyco-transfected with plasmids producing WT or mutant Nod1 and RICK. Theanalysis shows that the NH₂-terminal 1-648 amino acids, but not the LRRsof Nod1 mediates the interaction with RICK. The V41 Q point mutant inthe CARD, but not the K208R P-loop mutant, abolishs the association withRICK. In addition, another Nod-1 mutant (residues 126-953) fails toassociate with RICK. This result indicates that the CARD of Nod1 iscritical for the Nod1-RICK interaction. Reciprocal experiments revealthat the CARD, but not the kinase domain of RICK, is required for theassociation with Nod1. Thus, without intending to be bound, theseresults suggest that the Nod1/RICK interaction is mediated via theircorresponding CARDs.

[0931] Expression of Nod1 promotes both procaspase-9 activation andNF-kB activation and the latter may involve the association of Nod1 withRICK. To determine if the NF-kB-inducing activity of RICK requires Nod1or Caspase-9 activity, 293T cells are co-transfected with plasmidsexpressing RICK and mutant forms of Nod1 or catalytically inactiveCaspase-9 or Caspase-3 (as a control). Expression of Nod mutants lackingthe CARD (residues 126-953) or containing only the LRRs (residues649-953) inhibits RICK-mediated NF-kB activation but not that induced bytumor necrosis factor α (TNF α) stimulation. The results suggest thatboth RICK and Nod1 activate a TNF-a-independent pathway of NF-kBactivation. In addition, the NF-kB-inducing activity of RICK isunaffected by mutant Caspase-9 or Caspase-3, but is inhibited by mutantI-kB. The latter result indicates that Caspase-9 activity is notrequired for Nod1 or RICK to activate NF-kB activation and furthersuggests that both TNFA and the RICK/Nod-1 signaling pathways use commondownstream components such as NIK, IKKs and I-kB for activation ofNF-kB.

[0932] The above examples using Nod1 polypeptides can similarly be usedto identify binding partners and determine the functional activity ofother apoptosis related polypeptides of the present invention.

[0933] Certain apoptosis related polynucleotides and polypeptides of thepresent invention, including antibodies, were disclosed in U.S.provisional application serial Nos. 60/126,018, 60/139,638, and601,149,449, as well as in International Application No. PCT/US00/06642,each of which is herein incorporated by reference in its entirety.

[0934] It will be clear that the invention may be practiced otherwisethan as particularly described in the foregoing description andexamples. Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, are withinthe scope of the appended claims.

[0935] The entire disclosure of each document cited (including patents,patent applications, journal articles, abstracts, laboratory manuals,books, or other disclosures) in the Background of the Invention,Detailed Description, and Examples is hereby incorporated herein byreference. Further, the hard copy of the sequence listing submittedherewith and the corresponding computer readable form are bothincorporated herein by reference in their entireties.

1 27 1 733 DNA Homo sapiens 1 gggatccgga gcccaaatct tctgacaaaactcacacatg cccaccgtgc ccagcacctg 60 aattcgaggg tgcaccgtca gtcttcctcttccccccaaa acccaaggac accctcatga 120 tctcccggac tcctgaggtc acatgcgtggtggtggacgt aagccacgaa gaccctgagg 180 tcaagttcaa ctggtacgtg gacggcgtggaggtgcataa tgccaagaca aagccgcggg 240 aggagcagta caacagcacg taccgtgtggtcagcgtcct caccgtcctg caccaggact 300 ggctgaatgg caaggagtac aagtgcaaggtctccaacaa agccctccca acccccatcg 360 agaaaaccat ctccaaagcc aaagggcagccccgagaacc acaggtgtac accctgcccc 420 catcccggga tgagctgacc aagaaccaggtcagcctgac ctgcctggtc aaaggcttct 480 atccaagcga catcgccgtg gagtgggagagcaatgggca gccggagaac aactacaaga 540 ccacgcctcc cgtgctggac tccgacggctccttcttcct ctacagcaag ctcaccgtgg 600 acaagagcag gtggcagcag gggaacgtcttctcatgctc cgtgatgcat gaggctctgc 660 acaaccacta cacgcagaag agcctctccctgtctccggg taaatgagtg cgacggccgc 720 gactctagag gat 733 2 2045 DNA Homosapiens 2 cccgggtcga cccacgcgtc cggaaagatc caaaacaagt ggctgcggccgtcgcccagg 60 agtcatcgga cgccagaatc tggccgggtt ctgagcttgt tccgcctccctcccccggga 120 atggcgctat ccgggtcgac cccggccccg tgctgggagg aggatgagtgcctggactac 180 tacgggatgc tgtcgcttca ccgtatgttc gaggtggtgg gcgggcaactgaccgagtgc 240 gagctggagc tcctggcctt tctgctggat gaggctcctg gcgccgccggaggcttagcc 300 cgggcccgca gcggcctaga gctcctgctg gagctggagc gccgcgggcagtgcgacgag 360 agcaacctgc ggctgctggg gcaactcctg cgcgtgctgg cccgccacgacctgctgccg 420 cacctggcgc gcaagcggcg ccggccagtg tctccagaac gctatagctatggcacctcc 480 agctcttcaa agaggacaga gggtagctgc cgtcgccgtc ggcagtcaagcagttctgca 540 aattctcagc agggtcagtg ggagacaggc tcccccccaa ccaagcggcagcggcggagt 600 cggggccggc ccagtggtgg tgccagacgg cggcggagag gggccccagccgcaccccag 660 cagcagtcag agcccgccag accttcctct gaaggcaaag tgacctgtgacatccggctc 720 cgggttcgag cagagtactg cgagcatggg ccagccttgg agcagggcgtggcatcccgg 780 cggccccagg cgctggcgcg gcagctggac gtgtttgggc aggccaccgcagtgctgcgc 840 tcaagggacc tgggctctgt ggtttgtgac atcaagttct cagagctctcctatctggac 900 gccttctggg gcgactacct gagtggcgcc ctgctgcagg ccctgcggggcgtgttcctg 960 actgaggccc tgcgagaggc tgtgggccgg gaggctgttc gcctgctggtcagtgtggat 1020 gaggctgact atgaggctgg ccggcgccgc ctgttgctga tggaggaggaaggggggcgg 1080 cgcccgacag aggcctcctg atccaggact ggcaggattg atcccacctccaagtctccg 1140 ggccaccttc tcctgggagg acgaccatct ctacccctag aggactgtcactctagcatc 1200 tttgaggact gcgacaggac cgggacagca ggccccttga cagcccctcccacaggatgt 1260 gggctctgag gcctaaacca tttccagctg agtttccttc ccagactcctcctaccccca 1320 ggtgtgcccc cttagcctcc ggaggcgggg gctgggcctg tatctcagaagggaggggca 1380 cagctacaca ctcaccaaag gcccccctgc acattgtatc tctgatcttgggctgtctgc 1440 actgtcacag gtgcacacac tcgctcatgc tcacactgcc cctgctgagatcttccctgg 1500 gcctctgccc tggcctgctt cccagcacac acttctttgg cctaagggcttctctctcag 1560 gacctctaat ttgaccacaa ccaacctggg cttcagccac atcagtgggcactggagctg 1620 gggtgcacat ggggcctgct caccttgccc acacatctcc agccagccagggccctgccc 1680 agcttcaatt tacagacctg actctcctca ccttcccccc tgctgtccagagctgaacat 1740 agacttgcac ttggatgtca cctggagtgt cacatgggag tgttatggcagcatcatacc 1800 aaggcctact gttgcacatg gggccaaaac cagtaaacag ccaccttcttggaaagggaa 1860 tgcaaaggct ttgggggtga tggaaaagac ctttaacaaa tgataccaattaaactgccc 1920 tggaaagggc ataggtggga aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa 1980 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa 2040 aaaaa 2045 3 4415 DNA Homo sapiens 3 ctctagctctcagcggctgc gaagtctgta aacctggtgg ccaagtgatt gtaagtcagg 60 agactttccttcggtttctg cctttgatgg caatttcctt cggtttctgc ctttgatggc 120 aagaggtggagattgtggcg gcgattacag agaacgtctg ggaagacaag ttgctgtttt 180 tatgggaatcgcaggcttgg aagagacaga agcaattcca gaaataaatt ggaaattgaa 240 gatttaaacaatgttgtttt aaaatattct aacttcaaag aatgatgcca gaaacttaaa 300 aaggggctgcgcagagtagc aggggccctg gagggcgcgg cctgaatcct gattgccctt 360 ctgctgagaggacacacgca gctgaagatg aatttgggaa aagtagccgc ttgctacttt 420 aactatggaagagcagggcc acagtgagat ggaaataatc ccatcagagt ctcaccccca 480 cattcaattactgaaaagca atcgggaact tctggtcact cacatccgca atactcagtg 540 tctggtggacaacttgctga agaatgacta cttctcggcc gaagatgcgg agattgtgtg 600 tgcctgccccacccagcctg acaaggtccg caaaattctg gacctggtac agagcaaggg 660 cgaggaggtgtccgagttct tcctctactt gctccagcaa ctcgcagatg cctacgtgga 720 cctcaggccttggctgctgg agatcggctt ctccccttcc ctgctcactc agagcaaagt 780 cgtggtcaacactgacccag tgagcaggta tacccagcag ctgcgacacc atctgggccg 840 tgactccaagttcgtgctgt gctatgccca gaaggaggag ctgctgctgg aggagatcta 900 catggacaccatcatggagc tggttggctt cagcaatgag agcctgggca gcctgaacag 960 cctggcctgcctcctggacc acaccaccgg catcctcaat gagcagggtg agaccatctt 1020 catcctgggtgatgctgggg tgggcaagtc catgctgcta cagcggctgc agagcctctg 1080 ggccacgggccggctagacg caggggtcaa attcttcttc cactttcgct gccgcatgtt 1140 cagctgcttcaaggaaagtg acaggctgtg tctgcaggac ctgctcttca agcactactg 1200 ctacccagagcgggaccccg aggaggtgtt tgccttcctg ctgcgcttcc cccacgtggc 1260 cctcttcaccttcgatggcc tggacgagct gcactcggac ttggacctga gccgcgtgcc 1320 tgacagctcctgcccctggg agcctgccca ccccctggtc ttgctggcca acctgctcag 1380 tgggaagctgctcaaggggg ctagcaagct gctcacagcc cgcacaggca tcgaggtccc 1440 gcgccagttcctgcggaaga aggtgcttct ccggggcttc tcccccagcc acctgcgcgc 1500 ctatgccaggaggatgttcc ccgagcgggc cctgcaggac cgcctgctga gccagctgga 1560 ggccaaccccaacctctgca gcctgtgctc tgtgcccctc ttctgctgga tcatcttccg 1620 gtgcttccagcacttccgtg ctgcctttga aggctcacca cagctgcccg actgcacgat 1680 gaccctgacagatgtcttcc tcctggtcac tgaggtccat ctgaacagga tgcagcccag 1740 cagcctggtgcagcggaaca cacgcagccc agtggagacc ctccacgccg gccgggacac 1800 tctgtgctcgctggggcagg tggcccaccg gggcatggag aagagcctct ttgtcttcac 1860 ccaggaggaggtgcaggcct ccgggctgca ggagagagac atgcagctgg gcttcctgcg 1920 ggctttgccggagctgggcc ccgggggtga ccagcagtcc tatgagtttt tccacctcac 1980 cctccaggccttctttacag ccttcttcct cgtgctggac gacagggtgg gcactcagga 2040 gctgctcaggttcttccagg agtggatgcc ccctgcgggg gcagcgacca cgtcctgcta 2100 tcctcccttcctcccgttcc agtgcctgca gggcagtggt ccggcgcggg aagacctctt 2160 caagaacaaggatcacttcc agttcaccaa cctcttcctg tgcgggctgt tgtccaaagc 2220 caaacagaaactcctgcggc atctggtgcc cgcggcagcc ctgaggagaa agcgcaaggc 2280 cctgtgggcacacctgtttt ccagcctgcg gggctacctg aagagcctgc cccgcgttca 2340 ggtcgaaagcttcaaccagg tgcaggccat gcccacgttc atctggatgc tgcgctgcat 2400 ctacgagacacagagccaga aggtggggca gctggcggcc aggggcatct gcgccaacta 2460 cctcaagctgacctactgca acgcctgctc ggccgactgc agcgccctct ccttcgtcct 2520 gcatcacttccccaagcggc tggccctaga cctagacaac aacaatctca acgactacgg 2580 cgtgcgggagctgcagccct gcttcagccg cctcactgtt ctcagactca gcgtaaacca 2640 gatcactgacggtggggtaa aggtgctaag cgaagagctg accaaataca aaattgtgac 2700 ctatttgggtttatacaaca accagatcac cgatgtcgga gccaggtacg tcaccaaaat 2760 cctggatgaatgcaaaggcc tcacgcatct taaactggga aaaaacaaaa taacaagtga 2820 aggagggaagtatctcgccc tggctgtgaa gaacagcaaa tcaatctctg aggttgggat 2880 gtggggcaatcaagttgggg atgaaggagc aaaagccttc gcagaggctc tgcggaacca 2940 ccccagcttgaccaccctga gtcttgcgtc caacggcatc tccacagaag gaggaaagag 3000 ccttgcgagggccctgcagc agaacacgtc tctagaaata ctgtggctga cccaaaatga 3060 actcaacgatgaagtggcag agagtttggc agaaatgttg aaagtcaacc agacgttaaa 3120 gcatttatggcttatccaga atcagatcac agctaagggg actgcccagc tggcagatgc 3180 gttacagagcaacactggca taacagagat ttgcctaaat ggaaacctga taaaaccaga 3240 ggaggccaaagtctatgaag atgagaagcg gattatctgt ttctgagagg atgctttcct 3300 gttcatggggtttttgccct ggagcctcag cagcaaatgc cactctgggc agtcttttgt 3360 gtcagtgtcttaaaggggcc tgcgcaggcg ggactatcag gagtccactg cctccatgat 3420 gcaagccagcttcctgtgca gaaggtctgg tcggcaaact ccctaagtac ccgctacaat 3480 tctgcagaaaaagaatgtgt cttgcgagct gttgtagtta cagtaaatac actgtgaaga 3540 gactttattgcctattataa ttatttttat ctgaagctag aggaataaag ctgtgagcaa 3600 acagaggaggccagcctcac ctcattccaa cacctgccat agggaccaac gggagcgagt 3660 tggtcaccgctcttttcatt gaagagttga ggatgtggca caaagttggt gccaagcttc 3720 ttgaataaaacgtgtttgat ggattagtat tatacctgaa atattttctt ccttctcagc 3780 actttcccatgtattgatac tggtcccact tcacagctgg agacaccgga gtatgtgcag 3840 tgtgggatttgactcctcca aggttttgtg gaaagttaat gtcaaggaaa ggatgcacca 3900 cgggcttttaattttaatcc tggagtctca ctgtctgctg gcaaagatag agaatgccct 3960 cagctcttagctggtctaag aatgacgatg ccttcaaaat gctgcttcca ctcagggctt 4020 ctcctctgctaggctaccct cctctagaag gctgagtacc atgggctaca gtgtctggcc 4080 ttgggaagaagtgattctgt ccctccaaag aaatagggca tggcttgccc ctgtggccct 4140 ggcatccaaatggctgcttt tgtctccctt acctcgtgaa gaggggaagt ctcttcctgc 4200 ctcccaagcagctgaagggt gactaaacgg gcgccaagac tcaggggatc ggctgggaac 4260 tgggccagcagagcatgttg gacacccccc accatggtgg gcttgtggtg gctgctccat 4320 gagggtgggggtgatactac tagatcactt gtcctcttgc ccgctcattt gttaataaaa 4380 tactgaaaacaaaaaaaaaa aaaaaaaaaa aaaaa 4415 4 2066 DNA Homo sapiens 4 gcggcggcaaaatggcggcg cctgaggagc gggatctaac ccaggagcag acagagaagc 60 tgctgcagtttcaggatctc actggcatcg aatctatgga tcagtgtcgc cataccttgg 120 aacagcataactggaacata gaggctgctg tacaggacag attgaatgag caagagggcg 180 tacctagtgttttcaaccca cctccatcac gacccctgca ggttaataca gctgaccaca 240 ggatctacagctatgttgtc tcaagacctc aaccaagggg gctgcttgga tggggttatt 300 acttgataatgcttccattc cggtttacct attacacgat acttgatata tttaggtttg 360 ctcttcgttttatacggcct gaccctcgca gccgggtcac tgaccccgtt ggggacattg 420 tttcatttatgcactctttt gaagagaaat atgggagggc acaccctgtc ttctaccagg 480 gaacgtacagccaggcactt aacgatgcca aaagggagct tcgctttctt ttggtttatc 540 ttcatggagatgatcaccag gactctgatg agttttgtcg caacacactc tgtgcacctg 600 aagttatttcactaataaac actaggatgc tcttctgggc atgctctaca aacaaacctg 660 agggatacagggtctcacag gctttacgag agaacaccta tccattcctg gccatgatta 720 tgctgaaggatcgaaggatg actgtggtgg gacggctaga aggcctcatt caacctgatg 780 acctcattaaccaactgaca tttatcatgg atgctaacca gacttacctg gtgtcagaac 840 gcctagaaagggaagaaaga aaccagaccc aagtgctgag acaacagcag gatgaggcct 900 acctggcctctctcagagct gaccaggaga aagaaagaaa gaaacgggag gagcgggagc 960 gtaagcggcggaaggaggag gaggtgcaac agcaaaagtt ggcagaggag agacggcggc 1020 agaatttacaggaggaaaag gaaaggaagt tggaatgcct gccccctgaa ccttcccctg 1080 atgaccctgaaagtgtcaag atcatcttca aattacctaa tgattctcga gtagagagac 1140 gattccacttttcacagtct ctaacagtaa tccacgactt cttattctcc ttgaaggaaa 1200 gcccagaaaagtttcagatt gaagccaatt ttcccaggcg agtgctgccc tgcatccctt 1260 cagaggagtggcccaatccc cctacgctac aggaggccgg actcagccac acagaagttc 1320 tttttgttcaggacctaact gacgaatgac atttttttct tcctgtcccc tcctacccca 1380 gtccctaaaagaaatgggga aaaaagaaaa caacagcaag tcagaaaaaa aaaacaagag 1440 agagaaattcatattattat tattattata atacaatatt ttttttaaaa gactgctgca 1500 tccttaggaaggatcagaaa ccatgctgcc cgtaagagtc acaacctgtg tgtgcgcgca 1560 aggttagcaacaaacgtacc cgcttggcaa gcccaccctt cctgtggcct ctgtgcacgc 1620 accttccagtgaacagagac tcttcacctt cgacccatcc attgtcccag ctgggaaggg 1680 gacattcccactagttctca ttcattcttg cttttatgaa aaataaaagt gaaaaacctc 1740 catcaaccagctacttgcag catctcctga ggacttgctt ctcctgcctc tggggaagag 1800 agggaagagaaagcacagag cagagaagca gagatgttcc ttgaactgcc cacaagtttt 1860 caataacttttatttctgtt ttgtaatgac caaaggaatg aggctgacat aggtatatat 1920 atatatttttttcctttatt tgataaagag ccaattcttt aaacccatga gtttatgccc 1980 tgggctccttagcccacaat agtgtaataa aagtgccccg ggctggtttg tgcttaaaaa 2040 aaaaaaaaaaaaaaaaaaaa aaaaaa 2066 5 1406 DNA Homo sapiens 5 ggagcctcgt cgagtctccatgcaggtggt gcgcatggct ggccgagagc tgcaggccct 60 cctgggagaa ccagaggctgcagtgagccc cctgctgtgc ctgtcacaga gcggtccccc 120 cagcttcctc caaccggtcaccgtgcagct gcctctgccc tctggcatca caggcctcag 180 tctggaccgc tcccgcctgcacctgttgta ctgggcccct cctgcagcca cctgggatga 240 catcacagct caggtggtcctggagctcac ccacctgtac gcacgcttcc aggtactggc 300 tctggtacac caccaagaactgtgtgggag gcctggctcg gaaggcctgg gagcggctgc 360 ggctgcaccg tgtgaacctcatcgctctgc agcggcgccg ggaccctgag caggtcctgc 420 tgcagtgcct gccccgaaacaaggtggacg ccacccttcg gcggctgctg gagcggtacc 480 ggggccccga gccctctgacacggtggaga tgttcgaggg cgaagagttc tttgcggcct 540 tcgagcgcgg catygacgtggatgctgacc gccctgactg tgtggagggc agaatctgct 600 ttgtcttcta ctcgcacctgaagaatgtga aggaggtata cgtgaccacc actctggacc 660 gggaggctca ggctgtgcggggccaggtgt ccttctaccg tggcgcggtg cctgtgcggg 720 tgcccgagga ggctgaggctgcccggcaga ggaagggcgc agacgccctg tggatggcca 780 ctctgcccat caagctgccgagacttcgag ggtccgaggg gccacggcgg ggggctggcc 840 tctccttggc acccttgaatctgggagatg ccgagaccgg ctttctgacg cagagcarcc 900 tgctgartgt ggctgggcgtctgggtctgg actggccagc cgtggccctg cacctggggg 960 tgtcctaccg ggaggtgcagcgcatccggc acgagttccg ggatgatctg gatgagcaga 1020 tccgtcacat gctcttctcctgggctgagc gccaggctgg gcagccaggg gctgtggggc 1080 tcctggtgca ggccctggagcagagtgacc ggcaggacgt ggctgaagag gtgcgcgcag 1140 tcttggagct cggccgccgcaagtaccagg acagcatccg acgcatgggc ttggccccca 1200 aggaccccgc tctgcctggctcctcggctc cacagccccc agagcctgcc caggcctagg 1260 ccccacagac ttttaggctggcccagatat tccccagtgg atgggcagag cccccacctt 1320 caagtctctc cagtgtgtggggacgggtcc ctgtgagcaa caaaactgca ctgtttcttt 1380 caaaaaaaaa aaaaaaaaaaaactcc 1406 6 3172 DNA Homo sapiens SITE (148) n equals a,t,g, or c 6agcttcccgg gtgaggctgg gacccggcac cagggcagta ctgtggccgc tgcggcttca 60tctgccgact gggtcaggtt gcggagactc caggccgctt ccagggmgag tactcctgaa 120ttgtgaacat cacattcatc ccctgggncg atggagcttg tcactgagtg gaggcatcag 180ggggttggag ccttgtgaac agggaacctg ccccccaaca cttggaagga cctgggtttc 240agtgatgaga catggggtat gatgtaaccc gtttccaggg ggatgttgac gaagatctta 300tctgccctat ttgcagtgga gtcttggagg agccagtaca ggcacctcat tgtgaacatg 360ctttctgcaa cgcctgcatc acccagtggt tctctcagca acagacatgt ccagtggacc 420gtagtgttgt gacggtcgcc catctgcgcc cagtacctcg gatcatgcgg aacatgttgt 480caaagctgca gattgcctgt gacaacgctg tgttcggctg tagtgccgtt gtccggcttg 540acaacctcat gtctcacctc agcgactgtg agcacaaccc gaagcggcct gtgacctgtg 600aacagggctg tggcctggag atgcccaaag atgagctgcc caaccataac tgcattaagc 660acctgcgctc agtggtacag cagcagcaga cacgcatcgc agagctggag aagacgtcag 720ctgaacacaa acaccagctg gcggagcaga agcgagacat ccagctgcta aaggcataca 780tgcgtgcaat ccgcagtgtc aaccccaacc ttcagaacct ggaggagaca attgaataca 840acgagatcct agagtgggtg aactcccttc agccagcaag agtgacccgc tggggaggga 900tgatctcgac tcctgatgct gtgctccagg ctgtaatcaa gcgctccctg gtggagagtg 960gctgtcctgc ttctattgtc aacgagctga ttgaaaatgc ccacgagcgt agntggcccc 1020agggtctggc cacactagag actagacaga tgaaccgacg ctactatgag aactacgtgg 1080ccaagcgcat ccctggcaag caggctgttg tcgtgatggc ctgtgagaac cagcacatgg 1140gggatgacat ggtgcaagag ccaggccttg tcatgatatt tgcgcatggc gtggaagaga 1200tataagagaa ctcgactggc tatcaggaag agatggaaat cagaaaatcc catcactcca 1260gcagctggga cctgagtcct acccaccatt cttaatactg tggcttatac ctgagccaca 1320catctccctg cccttctggc actgaagggc cttggggtag tttgctcagc ctttcaggtg 1380ggaaacccag atttcctccc tttgccatat tcccctaaaa tgtctataaa ttatcagtct 1440gggtgggaaa gcccccacct ccatccattt tcctgcttag ggtccctggt tccagttatt 1500ttcagaaagc acaaagagat tcaatttccc tggaggatca ggacagagga aggaatctct 1560aatcgtccct ctcctccaaa accagggaat cagagcagtc aggcctgttg actctaagca 1620gcagacatcc tgaagaaatg gtaagggtgg agccaaatct ctagaaataa gtagtgaggc 1680cgttaattgg ccatcactga tggcccttag ggaaagactg gacctctgtg ccaagcagta 1740tccctgttca gcccacctta aaggtgtagg cacccactgg gtctaccagt atgcaggttg 1800ggatactgaa aatttccaga tgagctcttc tttcctacaa gttttcataa ttagggaatg 1860ccagggttta gggtaggggt taatctgttg ggggttgatg tgtttagcaa gaagctactc 1920ctagcttttg ctaaaatatg gttggcactg cctcttgtgg cacaggccat aattgttcca 1980tagacccctc tctagccctg tgactgtagt tagttacttt gataattttc tttggccatt 2040gtttgtttat atttcacaaa ctccacctac tgcccccccc cctctttttt ttaagaatgg 2100cctgatcatg gctatctcag ccacattgtt ggcaatttaa tttatttact tccttttttt 2160tttttaagaa aggaaaaaag aaaaaaaaat caaacttgaa acttttcttt tgakgttcct 2220attgtggggg ttctggatag ggtgggacag ggatgggggt gtgttttata ttttttcctt 2280ttcagcacaa cctttggctt taatatagga agagccaagg gagtcctcgg ctgaacttac 2340gatatctgcc ccaaacctct gtaaccccaa ctgaaatgag gagcttcctc tcttcctgtg 2400aaggatatga cagtccagca tcgatgcctg tgccctctgg aaaaatttcc tcctagccct 2460tccagggcct tatcataaaa ctctggattt agagtattca ttttgaaggc aactccccct 2520tccccaagtt tccttggagc tgtatagctg ggttctaagc ttcaccatgc aaatcagaaa 2580ttttatctct aagtacaggc tgtgccgtgt ctcacccaca cccccctggg gacttcagtt 2640ccatttcagg ttacctgggg tataccttga tccctagagt gactggcaga gtaagagaag 2700gggagagata ataggtgtga ttattttaat atggaggtgg gagtgtggtt ggagatagaa 2760aggctcctcc ccascatgta atggcttckc tcagaatttk attccaggct agcttgctsm 2820aggtctgggt agttggatca tgcgtcactg gaattggggg gaagcttgag gaagtaggtc 2880cagtctggaa atggctccga atttaaagct gatactagct ggtacgaatc ttcctccgtt 2940ccgagtgcgt acaggcataa gctgtcgtcc caggggccgg gagcgtaagt ccgcctgtcg 3000agcctcagga gcgggttgtg gtcagggtgt gggaaacagg tgtgcgtcct agaagaattg 3060gggctcttgg gtatggcggg gtaaggagat tgccaagtga ccctcggtta agccctcctg 3120gtggcgccgg gggtgggccc gaccttccta gggtcaaggc gctccccttc gg 3172 7 2290DNA Homo sapiens 7 gcccacgcgt ccgcccacgc gtccggatct ccaggacaatgaggttctta ctgagaatga 60 gaaggagctg gtggagcagg aaaagacacg gcagagcaagaatgaggcct tgctgagcat 120 ggtggagaag aaaggggacc tggccctgga cgtgctcttcagaagcatta gtgaaaggga 180 cccttacctc gtgtcctatc ttagacagca gaatttgtaaaatgagtcag ttaggtagtc 240 tggaagagag aatccagcgt tctcattgga aatggataaacagaaatgtg atcattgatt 300 tcagtgttca agacagaaga agactgggta acatctatcacacaggcttt caggacagac 360 ttgtaacctg gcatgtacct attgactgta tcctcatgcattttcctcaa gaatgtctga 420 agaaggtagt aatattcctt ttaaattttt tccaaccattgcttgatata tcactatttt 480 atccattgac atgattcttg aagacccagg ataaaggacatccggatagg tgtgtttatg 540 aaggatgggg cctggaaagg caacttttcc tgattaatgtgaaaaataat tcctatggac 600 actccgtttg aagtatcacc ttctcataac taaaagcagaaaagctaaca aaagcttctc 660 agctgaggac actcaaggca tacatgatga cagtctttttttttttttgt atgttaggac 720 tttaacactt tatctatggc tactgttatt agaacaatgtaaatgtattt gctgaaagag 780 agcacaaaaa tgggagaaaa tgcaaacatg agcagaaaatattttcccac tggtgtgtag 840 cctgctacaa ggagttgttg ggttaaatgt tcatggtcaactccaaggaa tactgagatg 900 aaatgtggta aatcaactcc acagaaccac caaaaagaaaatgagggtaa ttcagcttat 960 tctgagacag acattcctgg caatgtacca tacaaaaaataagccaactc tgacatttgg 1020 attctaccat agactctgtc attttgtagc catttcagctgtcttttgat taatgttttc 1080 gtggcacaca tatttccatc cttttatgtt taatctgtttaaaacaagtt cctagtagac 1140 accatctggt tgagtcagtt ttttttatgg tgtattttgaacccattctg atagtctctt 1200 ttaactggaa gatttcaatt acttacgtta atgtaattattaatatgtta ggatttatcc 1260 tcagtcagcc agtttgttat gtcttttcta ttctactgttatcacatttg taccacttaa 1320 agtggaatct aggcacttta tcaccattta gatcctattaccttttctca tctaggatat 1380 agttatcttc tacataatct ttctgtatct taaaacccatcaataaatta ttatatattt 1440 tctactttta atcactcaga agatttaaaa aactcatgagaagagtaatc tgttatgttt 1500 ttccagatat ttaccatttc tgttgctctt ccttcattattttccaaatt tcgttctgca 1560 aatttccact tcttctgata gacgtttttt agttcttttagagtggttct gataggtaca 1620 gattctctta ttttttgctt cctctgagga catctttttctcaccttcat tctcagtgat 1680 gttttttgct tgtagtattt ttagttgaca ttgttttctgttcagcagtt tccttttagc 1740 ttccgtattt cctgatgaga aatctgcagt cattcaaattgttgtttccc tgtatgtagt 1800 gtgtcatttt tctgtcagat ttcaaggtat ttatctttagtttttagcca tttcattatg 1860 ttggggatga gtttccttgt tttattccct ttggaatttgctccaattca taaatttgca 1920 gttttatgtc ttttaccaaa cttagaggtt ttcagcctaatttctaaaaa tactttttat 1980 tagcctgatt ttcatcttta taggaaatag tttaagtgatgacaagttcc aatagcttat 2040 atgcccagaa ggccttcaaa ataagaattt tgaaagaatacagaaaacaa acttttatat 2100 ccttctcatg tcttctactg taaaattcat atgctttgctactctaaacc tagtttgaaa 2160 tcaacagtct tgagaataga tgaaaatttt gatgaatagtggaattcttt taaatggaaa 2220 cctcttacat gtgattttcc ttgccatcta gaaataaaccatagtattta tgttgaaaaa 2280 aaaaaaaaaa 2290 8 1316 DNA Homo sapiens SITE(7) n equals a,t,g, or c 8 tcaattntca ggccagcgtg aggccatcac tgtgccgttcactgtgaaaa gaaaaccacc 60 ctggcgccca atacgcaaac cgcctctccc cgcgcgttggccgattcatt aatgcagctg 120 gcacgacagg tttcccgact ggaaagcggg cagtgagcgcaacgcaatta atgtgagtta 180 gctcactcat taggcacccc aggctttaca ctttatgcttccggctcgta tgttgtgtgg 240 aattgtgagc ggataacaat ttcacacagg aaacagctatgaccatgatt acgccaagct 300 cgaaattaac cctcactaaa gggaaagctg gtacgcctgcaggtaccggt ccggaattcc 360 cgggtcgacc cacgcgtccg cccacgcgtc cgtgaaaatccgaagtgccg cggaaagtgg 420 aggtgagggc cgcccgccct agaggtgccc gtccgagaggcagagctgac aaggaaggtt 480 tcgagcgttt tgctggcaaa gggatttctt acaacctccaggcatgcgtc tttctgccct 540 gctggccttg gcatccaagg tcactctgcc cccccattaccgctatggga tgagcccccc 600 aggctctgtt gcagacaaga ggaagaaccc cccatggatcaggcggcgcc cagtggttgt 660 ggaacccatc tctgatgaag actggtatct gttctgtggggacacggtgg agatcctaga 720 aggcaaggat gccgggaagc agggcaaagt ggttcaagttatccggcagc gaaactgggt 780 ggtcgtggga gggctgaaca cacattaccg ctacattggcaagaccatgg attaccgggg 840 aaccatgatc cctagtgaag cccccttgct ccaccgccaggtcaaacttg tggatcctat 900 ggacaggaaa cccactgaga tcgagtggag atttactgaagcaggagagc gggtacgagt 960 ctccacacga tcagggagaa ttatccctaa acccgaatttcccagagctg atggcatcgt 1020 ccctgaaacg tggattgatg gccccaaaga cacatcagtggaagatgctt tagaaagaac 1080 ctatgtgccc tgtctaaaga cactgcagga ggaggtgatggaggccatgg ggatcaagga 1140 gacccggaaa tacaagaagg tctattggta ttgagcctggggcagagcag ctcctcccca 1200 acttctgtcc cagccttgaa ggctgaggca cttctttttcagatgccaat aaagagcact 1260 ttatgagtca aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaa 1316 9 2150 DNA Homo sapiens 9 gcggacgcgt gggtcgacccacgcgtccgg tttgacccgt cggtcgtgcg tgagaggaaa 60 gggaaggagg aggtcccgaatagcggtcgc cgaaatgttc cggtgtggag gcctggcggc 120 gggtgctttg aagcagaagctggtgccctt ggtgcggacc gtgtgcgtcc gaagcccgag 180 gcagaggaac cggctcccaggcaacttgtt ccagcgatgg catgttcctc tagaactcca 240 gatgacaaga caaatggctagctctggtgc atcagggggc aaaatcgata attctgtgtt 300 agtccttatt gtgggcttatcaacagtagg agctggtgcc tatgcctaca agactatgaa 360 agaggatgaa aaaagatacaatgaaagaat ttcagggtta gggctgacac cagaacagaa 420 acagaaaaag gccgcgttatctgcttcaga aggagaggaa gttcctcaag acaaggcgcc 480 aagtcatgtt cctttcctgctaattggtgg aggcacagct gcttttgctg cagccagatc 540 catccgggct cgggatcctggggccagggt actgattgta tctgaagatc ctgagctgcc 600 gtacatgcga cctcctctttcaaaagaact gtggttttca gatgacccaa atgtcacaaa 660 gacactgcga ttcaaacagtggaatggaaa agagagaagc atatatttcc agccaccttc 720 tttctatgtc tctgctcaggacctgcctca tattgagaat ggtggtgtgg ctgtcctcac 780 tgggaagaag gtagtacagctggatgtgag agacaacatg gtgaaactta atgatggctc 840 tcaaataacc tatgaaaagtgcttgattgc aacaggaggt actccaagaa gtctgtctgc 900 cattgatagg gctggagcagaggtgaagag tagaacaacg cttttcagaa agattggaga 960 ctttagaagc ttggagaagatttcacggga agtcaaatca attacgatta tcggtggggg 1020 cttccttggt agcgaactggcctgtgctct tggcagaaag gctcgagcct tgggcacaga 1080 agtgattcaa ctcttccccgagaaaggaaa tatgggaaag atcctccccg aatacctcag 1140 caactggacc atggaaaaagtcagacgaga gggggttaag gtgatgccca atgctattgt 1200 gcaatccgtt ggagtcagcagtggcaagtt acttatcaag ctgaaagacg gcaggaaggt 1260 agaaactgac cacatagtggcagctgtggg cctggagccc aatgttgagt tggccaagac 1320 tggtggcctg gaaatagactcagattttgg tggcttccgg gtaaatgcag agctacaagc 1380 acgctctaac atctgggtggcaggagatgc tgcatgcttc tacgatataa agttgggaag 1440 gaggcgggta gagcaccatgatcacgctgt tgtgagtgga agattggctg gagaaaatat 1500 gactggagct gctaagccgtactggcatca gtcaatgttc tggagtgatt tgggccccga 1560 tgttggctat gaagctattggtcttgtgga cagtagtttg cccacagttg gtgtttttgc 1620 aaaagcaact gcacaagacaaccccaaatc tgccacagag cagtcaggaa ctggtatccg 1680 atcagagagt gagacagagtccgaggcctc agaaattact attcctccca gcaccccggc 1740 agttccacag gctcccgtccagggggagga ctacggcaaa ggtgtcatct tctacctcag 1800 ggacaaagtg gtcgtggggattgtgctatg gaacatcttt aaccgaatgc caatagcaag 1860 gaagatcatt aaggacggtgagcagcatga agatctcaat gaagtagcca aactattcaa 1920 cattcatgaa gactgaagccccacagtgga attggcaaac ccactgcagc ccctgagagg 1980 aggtcgaatg ggtaaaggagcattttttta ttcagcagac tttctctgtg tatgagtgtg 2040 aatgatcaag tcctttgtgaatattttcaa ctatgtaggt aaattcttaa tgttcacata 2100 gtgaaataaa ttctgattcttctaaaaaaa aaaaaaaaaa aaaaaaaaaa 2150 10 3789 DNA Homo sapiens SITE(3677) n equals a,t,g, or c 10 tcgacccacg cgtccgatcc catcagagtctcacccccac attcaattac tgaaaagcaa 60 tcgggaactt ctggtcactc acatccgcaatactcagtgt ctggtggaca acttgctgaa 120 gaatgactac ttctcggccg aagatgcggagattgtgtgt gcctgcccca cccagcctga 180 caaggtccgc aaaattctgg acctggtacagagcaagggc gaggaggtgt ccgagttctt 240 cctctacttg ctccagcaac tcgcagatgcctacgtggac ctcaggcctt ggctgctgga 300 gatcggcttc tccccttccc tgctcactcagagcaaagtc gtggtcaaca ctgacccagt 360 gagcaggtat acccagcagc tgcgacaccatctgggccgt gactccaagt tcgtgctgtg 420 ctatgcccag aaggaggagc tgctgctggaggagatctac atggacacca tcatggagct 480 ggttggcttc agcaatgaga gcctgggcagcctgaacagc ctggcctgcc tcctggacca 540 caccaccggc atcctcaatg agcagggacctgctcttcaa gcactactgc tacccagagc 600 gggaccccga ggaggtgttt gccttcctgctgcgcttccc ccacgtggcc ctcttcacct 660 tcgatggcct ggacgagctg cactcggacttggacctgag ccgcgtgcct gacagctcct 720 gcccctggga gcctgcccac cccctggtcttgctggccaa cctgctcagt gggaagctgc 780 tcaagggggc tagcaagctg ctcacagcccgcacaggcat cgaggtcccg cgccagttcc 840 tgcggaagaa ggtgcttctc cggggcttctcccccagcca cctgcgcgcc tatgccagga 900 ggatgttccc cgagcgggcc ctgcaggaccgcctgctgag ccagctggag gccaacccca 960 acctctgcag cctgtgctct gtgcccctcttctgctggat catcttccgg tgcttccagc 1020 acttccgtgc tgcctttgaa ggctcaccacagctgcccga ctgcacgatg accctgacag 1080 atgtcttcct cctggtcact gaggtccatctgaacaggat gcagcccagc agcctggtgc 1140 agcggaacac acgcagccca gtggagaccctccacgccgg ccgggacact ctgtgctcgc 1200 tggggcaggt ggcccaccgg ggcatggagaagagcctctt tgtcttcacc caggaggagg 1260 tgcakgcctc cgggctgcag gagagagacatgcagctggg cttyctgcgg gctttgccgg 1320 arctgggccc cggrggtgac cagcagtyctatgagttttt ccacctcacc ctccaggcct 1380 tctttacagc cttcttcctc gtgctggacgacagggtggg cactcaggag ctgctcaggt 1440 tcttccagga gtggatgccc cctgcgggggcagcgaccac gtcctgctat cctcccttcc 1500 tcccgttcca gtgcctgcag ggcagtggtccggcgcggga agacctcttc aagaacaagg 1560 atcacttcca gttcaccaac ctcttcctgtgcgggctgtt gtccaaagcc aaacagaaac 1620 tcctgcggca tctggtgccc gcggcagccctgaggagaaa gcgcaaggcc ctgtgggcac 1680 acctgttttc cagcctgcgg ggctacctgaagagcctgcc ccgcgttcag gtcgaaagct 1740 tcaaccaggt gcaggccatg cccacgttcatctggatgct gcgctgcatc tacgagacac 1800 agagccagaa ggtggggcag ctggcggccaggggcatctg cgccaactac ctcaagctga 1860 cctactgcaa cgcctgctcg gccgactgcagcgccctctc cttcgtcctg catcacttcc 1920 ccaagcggct ggccctagac ctagacaacaacaatctcaa cgactacggc gtgcgggagc 1980 tgcagccctg cttcagccgc ctcactgttctcagactcag cgtaaaccag atcactgacg 2040 gtggggtaaa ggtgctaagc gaagagctgaccaaatacaa aattgtgacc tatttgggtt 2100 tatacaacaa ccagatcacc gatgtcggagccaggtacgt caccaaaatc ctggatgaat 2160 gcaaaggcct cacgcatctt aaactgggaaaaaacaaaat aacaagtgaa ggagggaagt 2220 atctcgccct ggctgtgaag aacagcaaatcaatctctga ggttgggatg tggggcaatc 2280 aagttgggga tgaaggagca aaagccttcgcagaggctct gcggaaccac cccagcttga 2340 ccaccctgag tcttgcgtcc aacggcatctccacagaagg aggaaagagc cttgcgaggg 2400 ccctgcagca gaacacgtct ctagaaatactgtggctgac ccaaaatgaa ctcaamgatg 2460 aaktggcaga gagtttggca gaaatgttgaaagtcaacca gacgttaaag catttatggc 2520 ttatccagaa tcagatcaca gctaaggggactgcccagct ggcagatgcg ttacagagca 2580 acactggcat aacagagatt tgcctaaatggaaacctgat aaaaccagag gaggccaaag 2640 tctatgaaga tgagaagcgg attatctgtttctgagagga tgctttcctg ttcatggggt 2700 ttttgccctg gagcctcagc agcaaatgccactctgggca gtcttttgtg tcagtgtctt 2760 aaaggggcct gcgcaggcgg gactatcaggagtccactgc ctccatgatg caagccagct 2820 tcctgtgcag aaggtctggt cggcaaactccctaagtacc cgctacaatt ctgcagraaa 2880 agaatgtgtc ttgcgagctg ttgtagttacagtaaataca ctgtgaagag actttattgc 2940 ctattataat tatttttatc tgaagctagaggaataaagc tgtgagcaaa cagaggaggc 3000 cagcctcacc tcattccaac acctgccatagggaccaacg ggagcgagtt ggtcaccgct 3060 cttttcattg aagagttgag gatgtggcacaaagttggtg ccaagcttct tgaataaaac 3120 gtgtttgatg gattagtatt atacctgaaatattttcttc cttctcagca ctttcccatg 3180 tattgatact ggtcccactt cacagctggagacaccggag tatgtgcagt gtgggatttg 3240 actcctccaa ggttttgtgg aaagttaatgtcaaggaaag gatgcaccac gggcttttaa 3300 ttttaatcct ggagtctcac tgtctgctggcaaagataga gaatgccctc agctcttagc 3360 tggtctaaga atgacgatgc cttcaaaatgctgcttccac tcagggcttc tcctctgcta 3420 ggctaccctc ctctagaagg ctgagtaccatgggctacag tgtctggcct tgggaagaag 3480 tgattctgtc cctccaaaga aatagggcatggcttgcccc tgtggccctg gcatccaaat 3540 ggctgctttt gtctccctta cctcgtgaagaggggaagtc tcttcctgcc tcccaagcag 3600 ctgaagggtg actaaacggg cgccaagactcaggggatcg gctgggaact gggccagcag 3660 agcatgttgg acacccncca ccatggtgggcttgtggtgg ctgctccatg agggtggggg 3720 tgatactact agatcacttg tcctcttgccagctcatttg ttaataaaat actgaaaaca 3780 cwmaaaaaa 3789 11 304 PRT Homosapiens 11 Met Leu Ser Leu His Arg Met Phe Glu Val Val Gly Gly Gln LeuThr 1 5 10 15 Glu Cys Glu Leu Glu Leu Leu Ala Phe Leu Leu Asp Glu AlaPro Gly 20 25 30 Ala Ala Gly Gly Leu Ala Arg Ala Arg Ser Gly Leu Glu LeuLeu Leu 35 40 45 Glu Leu Glu Arg Arg Gly Gln Cys Asp Glu Ser Asn Leu ArgLeu Leu 50 55 60 Gly Gln Leu Leu Arg Val Leu Ala Arg His Asp Leu Leu ProHis Leu 65 70 75 80 Ala Arg Lys Arg Arg Arg Pro Val Ser Pro Glu Arg TyrSer Tyr Gly 85 90 95 Thr Ser Ser Ser Ser Lys Arg Thr Glu Gly Ser Cys ArgArg Arg Arg 100 105 110 Gln Ser Ser Ser Ser Ala Asn Ser Gln Gln Gly GlnTrp Glu Thr Gly 115 120 125 Ser Pro Pro Thr Lys Arg Gln Arg Arg Ser ArgGly Arg Pro Ser Gly 130 135 140 Gly Ala Arg Arg Arg Arg Arg Gly Ala ProAla Ala Pro Gln Gln Gln 145 150 155 160 Ser Glu Pro Ala Arg Pro Ser SerGlu Gly Lys Val Thr Cys Asp Ile 165 170 175 Arg Leu Arg Val Arg Ala GluTyr Cys Glu His Gly Pro Ala Leu Glu 180 185 190 Gln Gly Val Ala Ser ArgArg Pro Gln Ala Leu Ala Arg Gln Leu Asp 195 200 205 Val Phe Gly Gln AlaThr Ala Val Leu Arg Ser Arg Asp Leu Gly Ser 210 215 220 Val Val Cys AspIle Lys Phe Ser Glu Leu Ser Tyr Leu Asp Ala Phe 225 230 235 240 Trp GlyAsp Tyr Leu Ser Gly Ala Leu Leu Gln Ala Leu Arg Gly Val 245 250 255 PheLeu Thr Glu Ala Leu Arg Glu Ala Val Gly Arg Glu Ala Val Arg 260 265 270Leu Leu Val Ser Val Asp Glu Ala Asp Tyr Glu Ala Gly Arg Arg Arg 275 280285 Leu Leu Leu Met Glu Glu Glu Gly Gly Arg Arg Pro Thr Glu Ala Ser 290295 300 12 953 PRT Homo sapiens 12 Met Glu Glu Gln Gly His Ser Glu MetGlu Ile Ile Pro Ser Glu Ser 1 5 10 15 His Pro His Ile Gln Leu Leu LysSer Asn Arg Glu Leu Leu Val Thr 20 25 30 His Ile Arg Asn Thr Gln Cys LeuVal Asp Asn Leu Leu Lys Asn Asp 35 40 45 Tyr Phe Ser Ala Glu Asp Ala GluIle Val Cys Ala Cys Pro Thr Gln 50 55 60 Pro Asp Lys Val Arg Lys Ile LeuAsp Leu Val Gln Ser Lys Gly Glu 65 70 75 80 Glu Val Ser Glu Phe Phe LeuTyr Leu Leu Gln Gln Leu Ala Asp Ala 85 90 95 Tyr Val Asp Leu Arg Pro TrpLeu Leu Glu Ile Gly Phe Ser Pro Ser 100 105 110 Leu Leu Thr Gln Ser LysVal Val Val Asn Thr Asp Pro Val Ser Arg 115 120 125 Tyr Thr Gln Gln LeuArg His His Leu Gly Arg Asp Ser Lys Phe Val 130 135 140 Leu Cys Tyr AlaGln Lys Glu Glu Leu Leu Leu Glu Glu Ile Tyr Met 145 150 155 160 Asp ThrIle Met Glu Leu Val Gly Phe Ser Asn Glu Ser Leu Gly Ser 165 170 175 LeuAsn Ser Leu Ala Cys Leu Leu Asp His Thr Thr Gly Ile Leu Asn 180 185 190Glu Gln Gly Glu Thr Ile Phe Ile Leu Gly Asp Ala Gly Val Gly Lys 195 200205 Ser Met Leu Leu Gln Arg Leu Gln Ser Leu Trp Ala Thr Gly Arg Leu 210215 220 Asp Ala Gly Val Lys Phe Phe Phe His Phe Arg Cys Arg Met Phe Ser225 230 235 240 Cys Phe Lys Glu Ser Asp Arg Leu Cys Leu Gln Asp Leu LeuPhe Lys 245 250 255 His Tyr Cys Tyr Pro Glu Arg Asp Pro Glu Glu Val PheAla Phe Leu 260 265 270 Leu Arg Phe Pro His Val Ala Leu Phe Thr Phe AspGly Leu Asp Glu 275 280 285 Leu His Ser Asp Leu Asp Leu Ser Arg Val ProAsp Ser Ser Cys Pro 290 295 300 Trp Glu Pro Ala His Pro Leu Val Leu LeuAla Asn Leu Leu Ser Gly 305 310 315 320 Lys Leu Leu Lys Gly Ala Ser LysLeu Leu Thr Ala Arg Thr Gly Ile 325 330 335 Glu Val Pro Arg Gln Phe LeuArg Lys Lys Val Leu Leu Arg Gly Phe 340 345 350 Ser Pro Ser His Leu ArgAla Tyr Ala Arg Arg Met Phe Pro Glu Arg 355 360 365 Ala Leu Gln Asp ArgLeu Leu Ser Gln Leu Glu Ala Asn Pro Asn Leu 370 375 380 Cys Ser Leu CysSer Val Pro Leu Phe Cys Trp Ile Ile Phe Arg Cys 385 390 395 400 Phe GlnHis Phe Arg Ala Ala Phe Glu Gly Ser Pro Gln Leu Pro Asp 405 410 415 CysThr Met Thr Leu Thr Asp Val Phe Leu Leu Val Thr Glu Val His 420 425 430Leu Asn Arg Met Gln Pro Ser Ser Leu Val Gln Arg Asn Thr Arg Ser 435 440445 Pro Val Glu Thr Leu His Ala Gly Arg Asp Thr Leu Cys Ser Leu Gly 450455 460 Gln Val Ala His Arg Gly Met Glu Lys Ser Leu Phe Val Phe Thr Gln465 470 475 480 Glu Glu Val Gln Ala Ser Gly Leu Gln Glu Arg Asp Met GlnLeu Gly 485 490 495 Phe Leu Arg Ala Leu Pro Glu Leu Gly Pro Gly Gly AspGln Gln Ser 500 505 510 Tyr Glu Phe Phe His Leu Thr Leu Gln Ala Phe PheThr Ala Phe Phe 515 520 525 Leu Val Leu Asp Asp Arg Val Gly Thr Gln GluLeu Leu Arg Phe Phe 530 535 540 Gln Glu Trp Met Pro Pro Ala Gly Ala AlaThr Thr Ser Cys Tyr Pro 545 550 555 560 Pro Phe Leu Pro Phe Gln Cys LeuGln Gly Ser Gly Pro Ala Arg Glu 565 570 575 Asp Leu Phe Lys Asn Lys AspHis Phe Gln Phe Thr Asn Leu Phe Leu 580 585 590 Cys Gly Leu Leu Ser LysAla Lys Gln Lys Leu Leu Arg His Leu Val 595 600 605 Pro Ala Ala Ala LeuArg Arg Lys Arg Lys Ala Leu Trp Ala His Leu 610 615 620 Phe Ser Ser LeuArg Gly Tyr Leu Lys Ser Leu Pro Arg Val Gln Val 625 630 635 640 Glu SerPhe Asn Gln Val Gln Ala Met Pro Thr Phe Ile Trp Met Leu 645 650 655 ArgCys Ile Tyr Glu Thr Gln Ser Gln Lys Val Gly Gln Leu Ala Ala 660 665 670Arg Gly Ile Cys Ala Asn Tyr Leu Lys Leu Thr Tyr Cys Asn Ala Cys 675 680685 Ser Ala Asp Cys Ser Ala Leu Ser Phe Val Leu His His Phe Pro Lys 690695 700 Arg Leu Ala Leu Asp Leu Asp Asn Asn Asn Leu Asn Asp Tyr Gly Val705 710 715 720 Arg Glu Leu Gln Pro Cys Phe Ser Arg Leu Thr Val Leu ArgLeu Ser 725 730 735 Val Asn Gln Ile Thr Asp Gly Gly Val Lys Val Leu SerGlu Glu Leu 740 745 750 Thr Lys Tyr Lys Ile Val Thr Tyr Leu Gly Leu TyrAsn Asn Gln Ile 755 760 765 Thr Asp Val Gly Ala Arg Tyr Val Thr Lys IleLeu Asp Glu Cys Lys 770 775 780 Gly Leu Thr His Leu Lys Leu Gly Lys AsnLys Ile Thr Ser Glu Gly 785 790 795 800 Gly Lys Tyr Leu Ala Leu Ala ValLys Asn Ser Lys Ser Ile Ser Glu 805 810 815 Val Gly Met Trp Gly Asn GlnVal Gly Asp Glu Gly Ala Lys Ala Phe 820 825 830 Ala Glu Ala Leu Arg AsnHis Pro Ser Leu Thr Thr Leu Ser Leu Ala 835 840 845 Ser Asn Gly Ile SerThr Glu Gly Gly Lys Ser Leu Ala Arg Ala Leu 850 855 860 Gln Gln Asn ThrSer Leu Glu Ile Leu Trp Leu Thr Gln Asn Glu Leu 865 870 875 880 Asn AspGlu Val Ala Glu Ser Leu Ala Glu Met Leu Lys Val Asn Gln 885 890 895 ThrLeu Lys His Leu Trp Leu Ile Gln Asn Gln Ile Thr Ala Lys Gly 900 905 910Thr Ala Gln Leu Ala Asp Ala Leu Gln Ser Asn Thr Gly Ile Thr Glu 915 920925 Ile Cys Leu Asn Gly Asn Leu Ile Lys Pro Glu Glu Ala Lys Val Tyr 930935 940 Glu Asp Glu Lys Arg Ile Ile Cys Phe 945 950 13 445 PRT Homosapiens 13 Met Ala Ala Pro Glu Glu Arg Asp Leu Thr Gln Glu Gln Thr GluLys 1 5 10 15 Leu Leu Gln Phe Gln Asp Leu Thr Gly Ile Glu Ser Met AspGln Cys 20 25 30 Arg His Thr Leu Glu Gln His Asn Trp Asn Ile Glu Ala AlaVal Gln 35 40 45 Asp Arg Leu Asn Glu Gln Glu Gly Val Pro Ser Val Phe AsnPro Pro 50 55 60 Pro Ser Arg Pro Leu Gln Val Asn Thr Ala Asp His Arg IleTyr Ser 65 70 75 80 Tyr Val Val Ser Arg Pro Gln Pro Arg Gly Leu Leu GlyTrp Gly Tyr 85 90 95 Tyr Leu Ile Met Leu Pro Phe Arg Phe Thr Tyr Tyr ThrIle Leu Asp 100 105 110 Ile Phe Arg Phe Ala Leu Arg Phe Ile Arg Pro AspPro Arg Ser Arg 115 120 125 Val Thr Asp Pro Val Gly Asp Ile Val Ser PheMet His Ser Phe Glu 130 135 140 Glu Lys Tyr Gly Arg Ala His Pro Val PheTyr Gln Gly Thr Tyr Ser 145 150 155 160 Gln Ala Leu Asn Asp Ala Lys ArgGlu Leu Arg Phe Leu Leu Val Tyr 165 170 175 Leu His Gly Asp Asp His GlnAsp Ser Asp Glu Phe Cys Arg Asn Thr 180 185 190 Leu Cys Ala Pro Glu ValIle Ser Leu Ile Asn Thr Arg Met Leu Phe 195 200 205 Trp Ala Cys Ser ThrAsn Lys Pro Glu Gly Tyr Arg Val Ser Gln Ala 210 215 220 Leu Arg Glu AsnThr Tyr Pro Phe Leu Ala Met Ile Met Leu Lys Asp 225 230 235 240 Arg ArgMet Thr Val Val Gly Arg Leu Glu Gly Leu Ile Gln Pro Asp 245 250 255 AspLeu Ile Asn Gln Leu Thr Phe Ile Met Asp Ala Asn Gln Thr Tyr 260 265 270Leu Val Ser Glu Arg Leu Glu Arg Glu Glu Arg Asn Gln Thr Gln Val 275 280285 Leu Arg Gln Gln Gln Asp Glu Ala Tyr Leu Ala Ser Leu Arg Ala Asp 290295 300 Gln Glu Lys Glu Arg Lys Lys Arg Glu Glu Arg Glu Arg Lys Arg Arg305 310 315 320 Lys Glu Glu Glu Val Gln Gln Gln Lys Leu Ala Glu Glu ArgArg Arg 325 330 335 Gln Asn Leu Gln Glu Glu Lys Glu Arg Lys Leu Glu CysLeu Pro Pro 340 345 350 Glu Pro Ser Pro Asp Asp Pro Glu Ser Val Lys IleIle Phe Lys Leu 355 360 365 Pro Asn Asp Ser Arg Val Glu Arg Arg Phe HisPhe Ser Gln Ser Leu 370 375 380 Thr Val Ile His Asp Phe Leu Phe Ser LeuLys Glu Ser Pro Glu Lys 385 390 395 400 Phe Gln Ile Glu Ala Asn Phe ProArg Arg Val Leu Pro Cys Ile Pro 405 410 415 Ser Glu Glu Trp Pro Asn ProPro Thr Leu Gln Glu Ala Gly Leu Ser 420 425 430 His Thr Glu Val Leu PheVal Gln Asp Leu Thr Asp Glu 435 440 445 14 340 PRT Homo sapiens SITE(221) Xaa equals any of the naturally occurring L-amino acids 14 Met ThrSer Gln Leu Arg Trp Ser Trp Ser Ser Pro Thr Cys Thr His 1 5 10 15 AlaSer Arg Tyr Trp Leu Trp Tyr Thr Thr Lys Asn Cys Val Gly Gly 20 25 30 LeuAla Arg Lys Ala Trp Glu Arg Leu Arg Leu His Arg Val Asn Leu 35 40 45 IleAla Leu Gln Arg Arg Arg Asp Pro Glu Gln Val Leu Leu Gln Cys 50 55 60 LeuPro Arg Asn Lys Val Asp Ala Thr Leu Arg Arg Leu Leu Glu Arg 65 70 75 80Tyr Arg Gly Pro Glu Pro Ser Asp Thr Val Glu Met Phe Glu Gly Glu 85 90 95Glu Phe Phe Ala Ala Phe Glu Arg Gly Ile Asp Val Asp Ala Asp Arg 100 105110 Pro Asp Cys Val Glu Gly Arg Ile Cys Phe Val Phe Tyr Ser His Leu 115120 125 Lys Asn Val Lys Glu Val Tyr Val Thr Thr Thr Leu Asp Arg Glu Ala130 135 140 Gln Ala Val Arg Gly Gln Val Ser Phe Tyr Arg Gly Ala Val ProVal 145 150 155 160 Arg Val Pro Glu Glu Ala Glu Ala Ala Arg Gln Arg LysGly Ala Asp 165 170 175 Ala Leu Trp Met Ala Thr Leu Pro Ile Lys Leu ProArg Leu Arg Gly 180 185 190 Ser Glu Gly Pro Arg Arg Gly Ala Gly Leu SerLeu Ala Pro Leu Asn 195 200 205 Leu Gly Asp Ala Glu Thr Gly Phe Leu ThrGln Ser Xaa Leu Leu Xaa 210 215 220 Val Ala Gly Arg Leu Gly Leu Asp TrpPro Ala Val Ala Leu His Leu 225 230 235 240 Gly Val Ser Tyr Arg Glu ValGln Arg Ile Arg His Glu Phe Arg Asp 245 250 255 Asp Leu Asp Glu Gln IleArg His Met Leu Phe Ser Trp Ala Glu Arg 260 265 270 Gln Ala Gly Gln ProGly Ala Val Gly Leu Leu Val Gln Ala Leu Glu 275 280 285 Gln Ser Asp ArgGln Asp Val Ala Glu Glu Val Arg Ala Val Leu Glu 290 295 300 Leu Gly ArgArg Lys Tyr Gln Asp Ser Ile Arg Arg Met Gly Leu Ala 305 310 315 320 ProLys Asp Pro Ala Leu Pro Gly Ser Ser Ala Pro Gln Pro Pro Glu 325 330 335Pro Ala Gln Ala 340 15 317 PRT Homo sapiens SITE (254) Xaa equals any ofthe naturally occurring L-amino acids 15 Met Gly Tyr Asp Val Thr Arg PheGln Gly Asp Val Asp Glu Asp Leu 1 5 10 15 Ile Cys Pro Ile Cys Ser GlyVal Leu Glu Glu Pro Val Gln Ala Pro 20 25 30 His Cys Glu His Ala Phe CysAsn Ala Cys Ile Thr Gln Trp Phe Ser 35 40 45 Gln Gln Gln Thr Cys Pro ValAsp Arg Ser Val Val Thr Val Ala His 50 55 60 Leu Arg Pro Val Pro Arg IleMet Arg Asn Met Leu Ser Lys Leu Gln 65 70 75 80 Ile Ala Cys Asp Asn AlaVal Phe Gly Cys Ser Ala Val Val Arg Leu 85 90 95 Asp Asn Leu Met Ser HisLeu Ser Asp Cys Glu His Asn Pro Lys Arg 100 105 110 Pro Val Thr Cys GluGln Gly Cys Gly Leu Glu Met Pro Lys Asp Glu 115 120 125 Leu Pro Asn HisAsn Cys Ile Lys His Leu Arg Ser Val Val Gln Gln 130 135 140 Gln Gln ThrArg Ile Ala Glu Leu Glu Lys Thr Ser Ala Glu His Lys 145 150 155 160 HisGln Leu Ala Glu Gln Lys Arg Asp Ile Gln Leu Leu Lys Ala Tyr 165 170 175Met Arg Ala Ile Arg Ser Val Asn Pro Asn Leu Gln Asn Leu Glu Glu 180 185190 Thr Ile Glu Tyr Asn Glu Ile Leu Glu Trp Val Asn Ser Leu Gln Pro 195200 205 Ala Arg Val Thr Arg Trp Gly Gly Met Ile Ser Thr Pro Asp Ala Val210 215 220 Leu Gln Ala Val Ile Lys Arg Ser Leu Val Glu Ser Gly Cys ProAla 225 230 235 240 Ser Ile Val Asn Glu Leu Ile Glu Asn Ala His Glu ArgXaa Trp Pro 245 250 255 Gln Gly Leu Ala Thr Leu Glu Thr Arg Gln Met AsnArg Arg Tyr Tyr 260 265 270 Glu Asn Tyr Val Ala Lys Arg Ile Pro Gly LysGln Ala Val Val Val 275 280 285 Met Ala Cys Glu Asn Gln His Met Gly AspAsp Met Val Gln Glu Pro 290 295 300 Gly Leu Val Met Ile Phe Ala His GlyVal Glu Glu Ile 305 310 315 16 90 PRT Homo sapiens 16 Met Ser Gln LeuGly Ser Leu Glu Glu Arg Ile Gln Arg Ser His Trp 1 5 10 15 Lys Trp IleAsn Arg Asn Val Ile Ile Asp Phe Ser Val Gln Asp Arg 20 25 30 Arg Arg LeuGly Asn Ile Tyr His Thr Gly Phe Gln Asp Arg Leu Val 35 40 45 Thr Trp HisVal Pro Ile Asp Cys Ile Leu Met His Phe Pro Gln Glu 50 55 60 Cys Leu LysLys Val Val Ile Phe Leu Leu Asn Phe Phe Gln Pro Leu 65 70 75 80 Leu AspIle Ser Leu Phe Tyr Pro Leu Thr 85 90 17 216 PRT Homo sapiens 17 Met ArgLeu Ser Ala Leu Leu Ala Leu Ala Ser Lys Val Thr Leu Pro 1 5 10 15 ProHis Tyr Arg Tyr Gly Met Ser Pro Pro Gly Ser Val Ala Asp Lys 20 25 30 ArgLys Asn Pro Pro Trp Ile Arg Arg Arg Pro Val Val Val Glu Pro 35 40 45 IleSer Asp Glu Asp Trp Tyr Leu Phe Cys Gly Asp Thr Val Glu Ile 50 55 60 LeuGlu Gly Lys Asp Ala Gly Lys Gln Gly Lys Val Val Gln Val Ile 65 70 75 80Arg Gln Arg Asn Trp Val Val Val Gly Gly Leu Asn Thr His Tyr Arg 85 90 95Tyr Ile Gly Lys Thr Met Asp Tyr Arg Gly Thr Met Ile Pro Ser Glu 100 105110 Ala Pro Leu Leu His Arg Gln Val Lys Leu Val Asp Pro Met Asp Arg 115120 125 Lys Pro Thr Glu Ile Glu Trp Arg Phe Thr Glu Ala Gly Glu Arg Val130 135 140 Arg Val Ser Thr Arg Ser Gly Arg Ile Ile Pro Lys Pro Glu PhePro 145 150 155 160 Arg Ala Asp Gly Ile Val Pro Glu Thr Trp Ile Asp GlyPro Lys Asp 165 170 175 Thr Ser Val Glu Asp Ala Leu Glu Arg Thr Tyr ValPro Cys Leu Lys 180 185 190 Thr Leu Gln Glu Glu Val Met Glu Ala Met GlyIle Lys Glu Thr Arg 195 200 205 Lys Tyr Lys Lys Val Tyr Trp Tyr 210 21518 613 PRT Homo sapiens 18 Met Phe Arg Cys Gly Gly Leu Ala Ala Gly AlaLeu Lys Gln Lys Leu 1 5 10 15 Val Pro Leu Val Arg Thr Val Cys Val ArgSer Pro Arg Gln Arg Asn 20 25 30 Arg Leu Pro Gly Asn Leu Phe Gln Arg TrpHis Val Pro Leu Glu Leu 35 40 45 Gln Met Thr Arg Gln Met Ala Ser Ser GlyAla Ser Gly Gly Lys Ile 50 55 60 Asp Asn Ser Val Leu Val Leu Ile Val GlyLeu Ser Thr Val Gly Ala 65 70 75 80 Gly Ala Tyr Ala Tyr Lys Thr Met LysGlu Asp Glu Lys Arg Tyr Asn 85 90 95 Glu Arg Ile Ser Gly Leu Gly Leu ThrPro Glu Gln Lys Gln Lys Lys 100 105 110 Ala Ala Leu Ser Ala Ser Glu GlyGlu Glu Val Pro Gln Asp Lys Ala 115 120 125 Pro Ser His Val Pro Phe LeuLeu Ile Gly Gly Gly Thr Ala Ala Phe 130 135 140 Ala Ala Ala Arg Ser IleArg Ala Arg Asp Pro Gly Ala Arg Val Leu 145 150 155 160 Ile Val Ser GluAsp Pro Glu Leu Pro Tyr Met Arg Pro Pro Leu Ser 165 170 175 Lys Glu LeuTrp Phe Ser Asp Asp Pro Asn Val Thr Lys Thr Leu Arg 180 185 190 Phe LysGln Trp Asn Gly Lys Glu Arg Ser Ile Tyr Phe Gln Pro Pro 195 200 205 SerPhe Tyr Val Ser Ala Gln Asp Leu Pro His Ile Glu Asn Gly Gly 210 215 220Val Ala Val Leu Thr Gly Lys Lys Val Val Gln Leu Asp Val Arg Asp 225 230235 240 Asn Met Val Lys Leu Asn Asp Gly Ser Gln Ile Thr Tyr Glu Lys Cys245 250 255 Leu Ile Ala Thr Gly Gly Thr Pro Arg Ser Leu Ser Ala Ile AspArg 260 265 270 Ala Gly Ala Glu Val Lys Ser Arg Thr Thr Leu Phe Arg LysIle Gly 275 280 285 Asp Phe Arg Ser Leu Glu Lys Ile Ser Arg Glu Val LysSer Ile Thr 290 295 300 Ile Ile Gly Gly Gly Phe Leu Gly Ser Glu Leu AlaCys Ala Leu Gly 305 310 315 320 Arg Lys Ala Arg Ala Leu Gly Thr Glu ValIle Gln Leu Phe Pro Glu 325 330 335 Lys Gly Asn Met Gly Lys Ile Leu ProGlu Tyr Leu Ser Asn Trp Thr 340 345 350 Met Glu Lys Val Arg Arg Glu GlyVal Lys Val Met Pro Asn Ala Ile 355 360 365 Val Gln Ser Val Gly Val SerSer Gly Lys Leu Leu Ile Lys Leu Lys 370 375 380 Asp Gly Arg Lys Val GluThr Asp His Ile Val Ala Ala Val Gly Leu 385 390 395 400 Glu Pro Asn ValGlu Leu Ala Lys Thr Gly Gly Leu Glu Ile Asp Ser 405 410 415 Asp Phe GlyGly Phe Arg Val Asn Ala Glu Leu Gln Ala Arg Ser Asn 420 425 430 Ile TrpVal Ala Gly Asp Ala Ala Cys Phe Tyr Asp Ile Lys Leu Gly 435 440 445 ArgArg Arg Val Glu His His Asp His Ala Val Val Ser Gly Arg Leu 450 455 460Ala Gly Glu Asn Met Thr Gly Ala Ala Lys Pro Tyr Trp His Gln Ser 465 470475 480 Met Phe Trp Ser Asp Leu Gly Pro Asp Val Gly Tyr Glu Ala Ile Gly485 490 495 Leu Val Asp Ser Ser Leu Pro Thr Val Gly Val Phe Ala Lys AlaThr 500 505 510 Ala Gln Asp Asn Pro Lys Ser Ala Thr Glu Gln Ser Gly ThrGly Ile 515 520 525 Arg Ser Glu Ser Glu Thr Glu Ser Glu Ala Ser Glu IleThr Ile Pro 530 535 540 Pro Ser Thr Pro Ala Val Pro Gln Ala Pro Val GlnGly Glu Asp Tyr 545 550 555 560 Gly Lys Gly Val Ile Phe Tyr Leu Arg AspLys Val Val Val Gly Ile 565 570 575 Val Leu Trp Asn Ile Phe Asn Arg MetPro Ile Ala Arg Lys Ile Ile 580 585 590 Lys Asp Gly Glu Gln His Glu AspLeu Asn Glu Val Ala Lys Leu Phe 595 600 605 Asn Ile His Glu Asp 610 19705 PRT Homo sapiens SITE (236) Xaa equals any of the naturallyoccurring L-amino acids 19 Met Ser Arg Asp Leu Leu Phe Lys His Tyr CysTyr Pro Glu Arg Asp 1 5 10 15 Pro Glu Glu Val Phe Ala Phe Leu Leu ArgPhe Pro His Val Ala Leu 20 25 30 Phe Thr Phe Asp Gly Leu Asp Glu Leu HisSer Asp Leu Asp Leu Ser 35 40 45 Arg Val Pro Asp Ser Ser Cys Pro Trp GluPro Ala His Pro Leu Val 50 55 60 Leu Leu Ala Asn Leu Leu Ser Gly Lys LeuLeu Lys Gly Ala Ser Lys 65 70 75 80 Leu Leu Thr Ala Arg Thr Gly Ile GluVal Pro Arg Gln Phe Leu Arg 85 90 95 Lys Lys Val Leu Leu Arg Gly Phe SerPro Ser His Leu Arg Ala Tyr 100 105 110 Ala Arg Arg Met Phe Pro Glu ArgAla Leu Gln Asp Arg Leu Leu Ser 115 120 125 Gln Leu Glu Ala Asn Pro AsnLeu Cys Ser Leu Cys Ser Val Pro Leu 130 135 140 Phe Cys Trp Ile Ile PheArg Cys Phe Gln His Phe Arg Ala Ala Phe 145 150 155 160 Glu Gly Ser ProGln Leu Pro Asp Cys Thr Met Thr Leu Thr Asp Val 165 170 175 Phe Leu LeuVal Thr Glu Val His Leu Asn Arg Met Gln Pro Ser Ser 180 185 190 Leu ValGln Arg Asn Thr Arg Ser Pro Val Glu Thr Leu His Ala Gly 195 200 205 ArgAsp Thr Leu Cys Ser Leu Gly Gln Val Ala His Arg Gly Met Glu 210 215 220Lys Ser Leu Phe Val Phe Thr Gln Glu Glu Val Xaa Ala Ser Gly Leu 225 230235 240 Gln Glu Arg Asp Met Gln Leu Gly Phe Leu Arg Ala Leu Pro Glu Leu245 250 255 Gly Pro Gly Gly Asp Gln Gln Xaa Tyr Glu Phe Phe His Leu ThrLeu 260 265 270 Gln Ala Phe Phe Thr Ala Phe Phe Leu Val Leu Asp Asp ArgVal Gly 275 280 285 Thr Gln Glu Leu Leu Arg Phe Phe Gln Glu Trp Met ProPro Ala Gly 290 295 300 Ala Ala Thr Thr Ser Cys Tyr Pro Pro Phe Leu ProPhe Gln Cys Leu 305 310 315 320 Gln Gly Ser Gly Pro Ala Arg Glu Asp LeuPhe Lys Asn Lys Asp His 325 330 335 Phe Gln Phe Thr Asn Leu Phe Leu CysGly Leu Leu Ser Lys Ala Lys 340 345 350 Gln Lys Leu Leu Arg His Leu ValPro Ala Ala Ala Leu Arg Arg Lys 355 360 365 Arg Lys Ala Leu Trp Ala HisLeu Phe Ser Ser Leu Arg Gly Tyr Leu 370 375 380 Lys Ser Leu Pro Arg ValGln Val Glu Ser Phe Asn Gln Val Gln Ala 385 390 395 400 Met Pro Thr PheIle Trp Met Leu Arg Cys Ile Tyr Glu Thr Gln Ser 405 410 415 Gln Lys ValGly Gln Leu Ala Ala Arg Gly Ile Cys Ala Asn Tyr Leu 420 425 430 Lys LeuThr Tyr Cys Asn Ala Cys Ser Ala Asp Cys Ser Ala Leu Ser 435 440 445 PheVal Leu His His Phe Pro Lys Arg Leu Ala Leu Asp Leu Asp Asn 450 455 460Asn Asn Leu Asn Asp Tyr Gly Val Arg Glu Leu Gln Pro Cys Phe Ser 465 470475 480 Arg Leu Thr Val Leu Arg Leu Ser Val Asn Gln Ile Thr Asp Gly Gly485 490 495 Val Lys Val Leu Ser Glu Glu Leu Thr Lys Tyr Lys Ile Val ThrTyr 500 505 510 Leu Gly Leu Tyr Asn Asn Gln Ile Thr Asp Val Gly Ala ArgTyr Val 515 520 525 Thr Lys Ile Leu Asp Glu Cys Lys Gly Leu Thr His LeuLys Leu Gly 530 535 540 Lys Asn Lys Ile Thr Ser Glu Gly Gly Lys Tyr LeuAla Leu Ala Val 545 550 555 560 Lys Asn Ser Lys Ser Ile Ser Glu Val GlyMet Trp Gly Asn Gln Val 565 570 575 Gly Asp Glu Gly Ala Lys Ala Phe AlaGlu Ala Leu Arg Asn His Pro 580 585 590 Ser Leu Thr Thr Leu Ser Leu AlaSer Asn Gly Ile Ser Thr Glu Gly 595 600 605 Gly Lys Ser Leu Ala Arg AlaLeu Gln Gln Asn Thr Ser Leu Glu Ile 610 615 620 Leu Trp Leu Thr Gln AsnGlu Leu Xaa Asp Glu Xaa Ala Glu Ser Leu 625 630 635 640 Ala Glu Met LeuLys Val Asn Gln Thr Leu Lys His Leu Trp Leu Ile 645 650 655 Gln Asn GlnIle Thr Ala Lys Gly Thr Ala Gln Leu Ala Asp Ala Leu 660 665 670 Gln SerAsn Thr Gly Ile Thr Glu Ile Cys Leu Asn Gly Asn Leu Ile 675 680 685 LysPro Glu Glu Ala Lys Val Tyr Glu Asp Glu Lys Arg Ile Ile Cys 690 695 700Phe 705 20 40 PRT Homo sapiens 20 Pro Gly Ser Thr His Ala Ser Gly LysIle Gln Asn Lys Trp Leu Arg 1 5 10 15 Pro Ser Pro Arg Ser His Arg ThrPro Glu Ser Gly Arg Val Leu Ser 20 25 30 Leu Phe Arg Leu Pro Pro Pro Gly35 40 21 14 PRT Homo sapiens 21 Thr Ala Trp Pro Ala Ser Trp Thr Thr ProPro Ala Ser Ser 1 5 10 22 705 PRT Homo sapiens SITE (236) Xaa equals anyof the naturally occurring L-amino acids 22 Met Ser Arg Asp Leu Leu PheLys His Tyr Cys Tyr Pro Glu Arg Asp 1 5 10 15 Pro Glu Glu Val Phe AlaPhe Leu Leu Arg Phe Pro His Val Ala Leu 20 25 30 Phe Thr Phe Asp Gly LeuAsp Glu Leu His Ser Asp Leu Asp Leu Ser 35 40 45 Arg Val Pro Asp Ser SerCys Pro Trp Glu Pro Ala His Pro Leu Val 50 55 60 Leu Leu Ala Asn Leu LeuSer Gly Lys Leu Leu Lys Gly Ala Ser Lys 65 70 75 80 Leu Leu Thr Ala ArgThr Gly Ile Glu Val Pro Arg Gln Phe Leu Arg 85 90 95 Lys Lys Val Leu LeuArg Gly Phe Ser Pro Ser His Leu Arg Ala Tyr 100 105 110 Ala Arg Arg MetPhe Pro Glu Arg Ala Leu Gln Asp Arg Leu Leu Ser 115 120 125 Gln Leu GluAla Asn Pro Asn Leu Cys Ser Leu Cys Ser Val Pro Leu 130 135 140 Phe CysTrp Ile Ile Phe Arg Cys Phe Gln His Phe Arg Ala Ala Phe 145 150 155 160Glu Gly Ser Pro Gln Leu Pro Asp Cys Thr Met Thr Leu Thr Asp Val 165 170175 Phe Leu Leu Val Thr Glu Val His Leu Asn Arg Met Gln Pro Ser Ser 180185 190 Leu Val Gln Arg Asn Thr Arg Ser Pro Val Glu Thr Leu His Ala Gly195 200 205 Arg Asp Thr Leu Cys Ser Leu Gly Gln Val Ala His Arg Gly MetGlu 210 215 220 Lys Ser Leu Phe Val Phe Thr Gln Glu Glu Val Xaa Ala SerGly Leu 225 230 235 240 Gln Glu Arg Asp Met Gln Leu Gly Phe Leu Arg AlaLeu Pro Glu Leu 245 250 255 Gly Pro Gly Gly Asp Gln Gln Xaa Tyr Glu PhePhe His Leu Thr Leu 260 265 270 Gln Ala Phe Phe Thr Ala Phe Phe Leu ValLeu Asp Asp Arg Val Gly 275 280 285 Thr Gln Glu Leu Leu Arg Phe Phe GlnGlu Trp Met Pro Pro Ala Gly 290 295 300 Ala Ala Thr Thr Ser Cys Tyr ProPro Phe Leu Pro Phe Gln Cys Leu 305 310 315 320 Gln Gly Ser Gly Pro AlaArg Glu Asp Leu Phe Lys Asn Lys Asp His 325 330 335 Phe Gln Phe Thr AsnLeu Phe Leu Cys Gly Leu Leu Ser Lys Ala Lys 340 345 350 Gln Lys Leu LeuArg His Leu Val Pro Ala Ala Ala Leu Arg Arg Lys 355 360 365 Arg Lys AlaLeu Trp Ala His Leu Phe Ser Ser Leu Arg Gly Tyr Leu 370 375 380 Lys SerLeu Pro Arg Val Gln Val Glu Ser Phe Asn Gln Val Gln Ala 385 390 395 400Met Pro Thr Phe Ile Trp Met Leu Arg Cys Ile Tyr Glu Thr Gln Ser 405 410415 Gln Lys Val Gly Gln Leu Ala Ala Arg Gly Ile Cys Ala Asn Tyr Leu 420425 430 Lys Leu Thr Tyr Cys Asn Ala Cys Ser Ala Asp Cys Ser Ala Leu Ser435 440 445 Phe Val Leu His His Phe Pro Lys Arg Leu Ala Leu Asp Leu AspAsn 450 455 460 Asn Asn Leu Asn Asp Tyr Gly Val Arg Glu Leu Gln Pro CysPhe Ser 465 470 475 480 Arg Leu Thr Val Leu Arg Leu Ser Val Asn Gln IleThr Asp Gly Gly 485 490 495 Val Lys Val Leu Ser Glu Glu Leu Thr Lys TyrLys Ile Val Thr Tyr 500 505 510 Leu Gly Leu Tyr Asn Asn Gln Ile Thr AspVal Gly Ala Arg Tyr Val 515 520 525 Thr Lys Ile Leu Asp Glu Cys Lys GlyLeu Thr His Leu Lys Leu Gly 530 535 540 Lys Asn Lys Ile Thr Ser Glu GlyGly Lys Tyr Leu Ala Leu Ala Val 545 550 555 560 Lys Asn Ser Lys Ser IleSer Glu Val Gly Met Trp Gly Asn Gln Val 565 570 575 Gly Asp Glu Gly AlaLys Ala Phe Ala Glu Ala Leu Arg Asn His Pro 580 585 590 Ser Leu Thr ThrLeu Ser Leu Ala Ser Asn Gly Ile Ser Thr Glu Gly 595 600 605 Gly Lys SerLeu Ala Arg Ala Leu Gln Gln Asn Thr Ser Leu Glu Ile 610 615 620 Leu TrpLeu Thr Gln Asn Glu Leu Xaa Asp Glu Xaa Ala Glu Ser Leu 625 630 635 640Ala Glu Met Leu Lys Val Asn Gln Thr Leu Lys His Leu Trp Leu Ile 645 650655 Gln Asn Gln Ile Thr Ala Lys Gly Thr Ala Gln Leu Ala Asp Ala Leu 660665 670 Gln Ser Asn Thr Gly Ile Thr Glu Ile Cys Leu Asn Gly Asn Leu Ile675 680 685 Lys Pro Glu Glu Ala Lys Val Tyr Glu Asp Glu Lys Arg Ile IleCys 690 695 700 Phe 705 23 50 PRT Homo sapiens 23 Ala Pro Cys Cys AlaCys His Arg Ala Val Pro Pro Ala Ser Ser Asn 1 5 10 15 Arg Ser Pro CysSer Cys Leu Cys Pro Leu Ala Ser Gln Ala Ser Val 20 25 30 Trp Thr Ala ProAla Cys Thr Cys Cys Thr Gly Pro Leu Leu Gln Pro 35 40 45 Pro Gly 50 2434 PRT Homo sapiens 24 Ala Trp Trp Arg Arg Lys Gly Thr Trp Pro Trp ThrCys Ser Ser Glu 1 5 10 15 Ala Leu Val Lys Gly Thr Leu Thr Ser Cys ProIle Leu Asp Ser Ile 20 25 30 Cys Lys 25 18 PRT Homo sapiens 25 Gln GlyArg Phe Arg Ala Phe Cys Trp Gln Arg Asp Phe Leu Gln Pro 1 5 10 15 ProGly 26 613 PRT Homo sapiens 26 Met Phe Arg Cys Gly Gly Leu Ala Ala GlyAla Leu Lys Gln Lys Leu 1 5 10 15 Val Pro Leu Val Arg Thr Val Cys ValArg Ser Pro Arg Gln Arg Asn 20 25 30 Arg Leu Pro Gly Asn Leu Phe Gln ArgTrp His Val Pro Leu Glu Leu 35 40 45 Gln Met Thr Arg Gln Met Ala Ser SerGly Ala Ser Gly Gly Lys Ile 50 55 60 Asp Asn Ser Val Leu Val Leu Ile ValGly Leu Ser Thr Val Gly Ala 65 70 75 80 Gly Ala Tyr Ala Tyr Lys Thr MetLys Glu Asp Glu Lys Arg Tyr Asn 85 90 95 Glu Arg Ile Ser Gly Leu Gly LeuThr Pro Glu Gln Lys Gln Lys Lys 100 105 110 Ala Ala Leu Ser Ala Ser GluGly Glu Glu Val Pro Gln Asp Lys Ala 115 120 125 Pro Ser His Val Pro PheLeu Leu Ile Gly Gly Gly Thr Ala Ala Phe 130 135 140 Ala Ala Ala Arg SerIle Arg Ala Arg Asp Pro Gly Ala Arg Val Leu 145 150 155 160 Ile Val SerGlu Asp Pro Glu Leu Pro Tyr Met Arg Pro Pro Leu Ser 165 170 175 Lys GluLeu Trp Phe Ser Asp Asp Pro Asn Val Thr Lys Thr Leu Arg 180 185 190 PheLys Gln Trp Asn Gly Lys Glu Arg Ser Ile Tyr Phe Gln Pro Pro 195 200 205Ser Phe Tyr Val Ser Ala Gln Asp Leu Pro His Ile Glu Asn Gly Gly 210 215220 Val Ala Val Leu Thr Gly Lys Lys Val Val Gln Leu Asp Val Arg Asp 225230 235 240 Asn Met Val Lys Leu Asn Asp Gly Ser Gln Ile Thr Tyr Glu LysCys 245 250 255 Leu Ile Ala Thr Gly Gly Thr Pro Arg Ser Leu Ser Ala IleAsp Arg 260 265 270 Ala Gly Ala Glu Val Lys Ser Arg Thr Thr Leu Phe ArgLys Ile Gly 275 280 285 Asp Phe Arg Ser Leu Glu Lys Ile Ser Arg Glu ValLys Ser Ile Thr 290 295 300 Ile Ile Gly Gly Gly Phe Leu Gly Ser Glu LeuAla Cys Ala Leu Gly 305 310 315 320 Arg Lys Ala Arg Ala Leu Gly Thr GluVal Ile Gln Leu Phe Pro Glu 325 330 335 Lys Gly Asn Met Gly Lys Ile LeuPro Glu Tyr Leu Ser Asn Trp Thr 340 345 350 Met Glu Lys Val Arg Arg GluGly Val Lys Val Met Pro Asn Ala Ile 355 360 365 Val Gln Ser Val Gly ValSer Ser Gly Lys Leu Leu Ile Lys Leu Lys 370 375 380 Asp Gly Arg Lys ValGlu Thr Asp His Ile Val Ala Ala Val Gly Leu 385 390 395 400 Glu Pro AsnVal Glu Leu Ala Lys Thr Gly Gly Leu Glu Ile Asp Ser 405 410 415 Asp PheGly Gly Phe Arg Val Asn Ala Glu Leu Gln Ala Arg Ser Asn 420 425 430 IleTrp Val Ala Gly Asp Ala Ala Cys Phe Tyr Asp Ile Lys Leu Gly 435 440 445Arg Arg Arg Val Glu His His Asp His Ala Val Val Ser Gly Arg Leu 450 455460 Ala Gly Glu Asn Met Thr Gly Ala Ala Lys Pro Tyr Trp His Gln Ser 465470 475 480 Met Phe Trp Ser Asp Leu Gly Pro Asp Val Gly Tyr Glu Ala IleGly 485 490 495 Leu Val Asp Ser Ser Leu Pro Thr Val Gly Val Phe Ala LysAla Thr 500 505 510 Ala Gln Asp Asn Pro Lys Ser Ala Thr Glu Gln Ser GlyThr Gly Ile 515 520 525 Arg Ser Glu Ser Glu Thr Glu Ser Glu Ala Ser GluIle Thr Ile Pro 530 535 540 Pro Ser Thr Pro Ala Val Pro Gln Ala Pro ValGln Gly Glu Asp Tyr 545 550 555 560 Gly Lys Gly Val Ile Phe Tyr Leu ArgAsp Lys Val Val Val Gly Ile 565 570 575 Val Leu Trp Asn Ile Phe Asn ArgMet Pro Ile Ala Arg Lys Ile Ile 580 585 590 Lys Asp Gly Glu Gln His GluAsp Leu Asn Glu Val Ala Lys Leu Phe 595 600 605 Asn Ile His Glu Asp 61027 31 PRT Homo sapiens 27 Arg Thr Arg Gly Ser Thr His Ala Ser Gly LeuThr Arg Arg Ser Cys 1 5 10 15 Val Arg Gly Lys Gly Arg Arg Arg Ser ArgIle Ala Val Ala Glu 20 25 30

What is claimed is:
 1. An isolated nucleic acid molecule comprising apolynucleotide selected from the group consisting of: (a) thepolynucleotide shown as SEQ ID NO:X or the polynucleotide encoded by acDNA included in ATCC Deposit No:Z; (b) a polynucleotide encoding abiologically active polypeptide fragment of SEQ ID NO:Y or abiologically active polypeptide fragment encoded by the cDNA sequenceincluded in ATCC Deposit No:Z; (c) a polynucleotide encoding apolypeptide epitope of SEQ ID NO:Y or a polypeptide epitope encoded bythe cDNA sequence included in ATCC Deposit No:Z; (d) a polynucleotidecapable of hybridizing under stringent conditions to any one of thepolynucleotides specified in (a)-(c), wherein said polynucleotide doesnot hybridize under stringent conditions to a nucleic acid moleculehaving a nucleotide sequence of only A residues or of only T residues.2. The isolated nucleic acid molecule of claim 1, wherein thepolynucleotide comprises a nucleotide sequence encoding a solublepolypeptide.
 3. The isolated nucleic acid molecule of claim 1, whereinthe polynucleotide comprises a nucleotide sequence encoding the sequenceidentified as SEQ ID NO:Y or the polypeptide encoded by the cDNAsequence included in ATCC Deposit No:Z.
 4. The isolated nucleic acidmolecule of claim 1, wherein the polynucleotide comprises the entirenucleotide sequence of SEQ ID NO:X or a cDNA included in ATCC DepositNo:Z.
 5. The isolated nucleic acid molecule of claim 2, wherein thepolynucleotide is DNA.
 6. The isolated nucleic acid molecule of claim 3,wherein the polynucleotide is RNA.
 7. A vector comprising the isolatednucleic acid molecule of claim
 1. 8. A host cell comprising the vectorof claim
 7. 9. A recombinant host cell comprising the nucleic acidmolecule of claim 1 operably limited to a heterologous regulatingelement which controls gene expression.
 10. A method of producing apolypeptide comprising expressing the encoded polypeptide from the hostcell of claim 9 and recovering said polypeptide.
 11. An isolatedpolypeptide comprising an amino acid sequence at least 95% identical toa sequence selected from the group consisting of: (a) the polypeptideshown as SEQ iID NO:Y or the polypeptide encoded by the cDNA; (b) apolypeptide fragment of SEQ ID NO:Y or the polypeptide encoded by thecDNA; (c) a polypeptide epitope of SEQ ID NO:Y or the polypeptideencoded by the cDNA; and (d) a variant of SEQ ID NO:Y.
 12. The isolatedpolypeptide of claim 11, comprising a polypeptide having SEQ ID NO:Y.13. An isolated antibody that binds specifically to the isolatedpolypeptide of claim
 11. 14. A recombinant host cell that expresses theisolated polypeptide of claim
 11. 15. A method of making an isolatedpolypeptide comprising: (a) culturing the recombinant host cell of claim14 under conditions such that said polypeptide is expressed; and (b)recovering said polypeptide.
 16. The polypeptide produced by claim 15.17. A method for preventing, treating, or ameliorating a medicalcondition, comprising administering to a mammalian subject atherapeutically effective amount of the polypeptide of claim
 11. 18. Amethod of diagnosing a pathological condition or a susceptibility to apathological condition in a subject comprising: (a) determining thepresence or absence of a mutation in the polynucleotide of claim 1; and(b) diagnosing a pathological condition or a susceptibility to apathological condition based on the presence or absence of saidmutation.
 19. A method of diagnosing a pathological condition or asusceptibility to a pathological condition in a subject comprising: (a)determining the presence or amount of expression of the polypeptide ofclaim 11 in a biological sample; and (b) diagnosing a pathologicalcondition or a susceptibility to a pathological condition based on thepresence or amount of expression of the polypeptide.
 20. A method foridentifying a binding partner to the polypeptide of claim 11 comprising:(a) contacting the polypeptide of claim 11 with a binding partner; and(b) determining whether the binding partner effects an activity of thepolypeptide.
 21. A method of screening for molecules which modifyactivities of the polypeptide of claim 11 comprising: (a) contactingsaid polypeptide with a compound suspected of having agonist orantagonist activity; and (b) assaying for activity of said polypeptide.22. A method for preventing, treating, or ameliorating a medicalcondition, comprising administering to a mammalian subject atherapeutically effective amount of the polynucleotide of claim 1.